Tlr4-deficient mice harbored fewer parasites in their spleen and liver than wild-type mice. TLR4 deficiency enhanced the Th1 immune response against the parasite, which was correlated with an increased activation of dendritic cells (DCs). Gene expression analyses demonstrated that IRF1 and IFN-β were expressed downstream of TLR4 after infection. Accordingly, IRF1- and IFNAR-deficient mice harbored fewer parasites in the target organs than wild-type mice due to having an increased Th1 immune response. However, the absence of TLR4 or IFNAR increased the serum transaminase levels in infected mice, indicating the presence of liver damage in these animals. In addition, IFN-β limits IFN-γ production by acting directly on Th1 cells. Using RNA sequencing analysis of human samples, we demonstrated that the transcriptional signature for the TLR4 and type I IFN (IFN-I) pathways was positively modulated in asymptomatic subjects compared with VL patients and thus provide direct evidence demonstrating that the TLR4-IFN-I pathway is related to the nondevelopment of the disease. In conclusion, our results demonstrate that the TLR4-IRF1 pathway culminates in IFN-β production as a mechanism for dampening the chronic inflammatory process and preventing immunopathology development.A striking feature of human visceral leishmaniasis (VL) is chronic inflammation in the spleen and liver, and VL patients present increased production levels of multiple inflammatory mediators, which contribute to tissue damage and disease severity. Here, we combined an experimental model with the transcriptional profile of human VL to demonstrate that the TLR4-IFN-β pathway regulates the chronic inflammatory process and is associated with the asymptomatic form of the disease.  Leishmania parasites. Most subjects infected with Leishmania present subclinical VL symptoms, and their immune response is mediated by Th1 cells and immunoregulatory mechanisms. However, when infection progresses to disease, VL patients present increased levels of inflammatory mediators in the serum which are related to the severity of disease. During infection, Toll-like receptors (TLRs) interact with Leishmania parasites and contribute to the outcome of the disease. Herein, we report that TLR4 signaling hampers the chronic immune response during VL to prevent immunopathology. TLR4 triggers the activation of IRF1 and thus induces the transcription of IFN-β, which in turn acts directly on Th1 cells to limit the production of IFN-γ. In addition, a transcription analysis of human VL samples provides direct evidence demonstrating that the TLR4-IFN-I pathway is related to the asymptomatic form of the disease. Collectively, our findings reveal that TLR4 hampers the Th1 immune response through IRF1 and IFN-β to prevent immunopathology during VL.Visceral leishmaniasis (VL) is one of the most lethal neglected tropical diseases and is caused by  Leishmania spp. Among the clinical manifestations of this disease, visceral leishmaniasis (VL) is the most severe form and causes high human mortality and morbidity around the world. VL, which results from infection with Leishmania donovani and Leishmania infantum, affects mainly the visceral organs and induces a chronic and potentially fatal pathology if left untreated following clinical diagnosis.Leishmaniasis is a broad-spectrum disease caused by the intracellular protozoan parasite Leishmania species have evolved a wide variety of strategies to evade host immune mechanisms and thus maintain a chronic infectious state and survive within the host [Leishmania present subclinical VL symptoms and are considered asymptomatic because they exhibit a balance between effector mechanisms related to the development of a Th1-mediated immune response against Leishmania antigens and immunoregulatory mechanisms [the host ,2. Approchanisms . Once thchanisms . In addichanisms –8. This Leishmania infection, and these cells trigger an effective adaptive response due to their wide repertoire of pattern recognition receptors (PRRs) [Leishmania infection can hamper DC function through the manipulation of different pathways, such as inhibition of their migration to T cell-containing areas of the spleen due to a loss of CCR7 expression [L. infantum parasites infect and survive inside DCs [Host-parasite interactions during the innate immune response determine the fate of adaptive immunity and contribute to parasite persistence or replication control in leishmaniasis . In thiss (PRRs) ,11. Othepression , inhibitpression , inhibitpression  and IL-1pression . L. infaside DCs  and inhiside DCs .Leishmania species during the initial sensing of these parasites and mount the subsequent adaptive immune response [Toll-like receptors (TLRs) are thought to be among the most ancient pathogen recognition systems , and theresponse . Our groresponse ,21.Leishmania major infection [L. donovani and proteoglycolipid complex (P8GLC) from Leishmania pifanoi [Tlr4-/- mice infected with L. donovani exhibit deficient production of proinflammatory cytokines, which culminates in increased parasite replication in the target organs of the disease [L. donovani has devised mechanisms to alter the TLR4 signaling pathway to favor establishment of infection [L. infantum remains to be elucidated. We herein demonstrate that the TLR4 signaling pathway abrogates the Th1 immune response through an interferon regulatory factor 1 (IRF1)- and interferon alpha/beta receptor (IFNAR)-dependent mechanism to avoid tissue damage. In human samples, the gene signature for TLR4 and type I IFN (IFN-I) was found to be positively modulated in asymptomatic subjects compared with VL patients. Taken together, our results reveal the relevance of the TLR4-IRF1 pathway culminating in IFN-β production as a mechanism for attenuating the immune response and consequently preventing immunopathology development.TLR4 plays a role in the control of nfection ,23 and i pifanoi ,25. In t disease . Moreovenfection ,28. The Tlr4 expression in the target organs of L. infantum-infected wild-type (WT) C57BL/6 mice were evaluated. High levels of Tlr4 messenger RNA (mRNA) expression were detected initially after 48 h of infection and later at 4 and 6 weeks post-infection (wpi), in the spleen  and late (6 wpi). The percentage and total number of spleen CD11b+CD11chigh cells at 6 weeks after L. infantum infection were increased in both the WT and Tlr4-/- mice compared with the respective naïve group  infected with parasite. In these cells, IL-12p40 has been determined to be an essential cytokine for overcoming L. infantum infection through Th1 subset differentiation [L. infantum was blunted in the absence of MYD88   . Reinfored BMDCs .Tlr4-/- splenic DCs, we evaluated the statuses of the coinhibitory molecules PD-L1 and CTLA-4 in these cells. Both the percentages and median fluorescence intensities (MFIs) of these molecules in splenic DCs from the infected WT mice were similar to those in splenic DCs from the infected Tlr4-/- mice . Parasite infection induced significant mRNA expression in the spleen at 2 wpi and persisted for more than 3 wpi  and control 129/SvEv WT mice. Among all the time points analyzed, the Ifnar1-/- mice harbored fewer parasites in both target organs than the WT mice at 6 wpi , cultured under Th1 conditions in the presence or absence of recombinant IFN-β (rIFN-β) and then stimulated with α-CD3 and α-CD28 for 4 days. The proliferation of these cells was then analyzed by CFSE positivity, and Th1 differentiation was evaluated through intracellular staining of IFN-γ production or T-bet expression. In addition, the IFN-γ levels in the culture supernatant were measured. The addition of rIFN-β did not alter the frequency of CFSE-labeled CD4 T cells  or the e T cells  but did t+ cells  and the t+ cells  by approin vivo infection, we transferred CD4+ cells from Ifnar1-/- or C57BL/6 WT mice to Rag1-deficient mice (Rag1-/- mice). The transferred mice were then infected, and the production of IFN-γ by their splenic CD4+ T cells was assessed at 6 wpi. Direct ex vivo flow cytometric measurement of IFN-γ production revealed that Rag mice transferred with Ifnar1-/- CD4+ cells contained higher numbers of CD4+ T cells producing IFN-γ compared with those transferred with WT CD4+ cells . The comparison of the VL patients with the control subjects revealed that many transcripts related to the IFN-I (red bars) and TLR4 (blue bars) pathways were positively modulated in the asymptomatic patients and negatively modulated in the VL patients . In agreIn each comparison, a total of 46 differentially expressed genes (DEGs) matched the gene signature list for the IFN-I signaling pathway. By overlapping these DEGs  expression [P. chabaudi-infected mice, IFN-I suppresses Th1 responses by modulating DC functions [Ifnar-/- mice infected with T. gondii presented an increased parasitic load, and higher parasitic burdens correlated with decreased mouse survival [T. brucei infection, type I IFNs play a role in the early control of parasites but contribute to the downregulation of IFN-γ production in the chronic phase of infection [Our results demonstrated that IFN-β secretion is dependent on the TLR4-IRF1 pathway, which in turn directly mitigates Th1 immune responses. Indeed, IFNAR deficiency and specific IFNAR blockade improve the Th1 response to pression . Our datpression . The rolunctions . In contunctions . In addisurvival , suggestnfection .+ T-cell-dependent manner [Mycobacterium tuberculosis and Mycobacterium leprae infections, IFN-I suppresses Th1 cells through the induction of IL-10 expression [+ T-cell activation and reduces the viral burden in the blood [The modulatory function of IFN-I may be dependent upon the timing of production and upon specific pathogens, as IFNAR signaling assumes an immunosuppressive role to limit host toxicity when the microorganism cannot be cleared . We obset manner ,69. Duripression ,46. In ahe blood ,71. As VMKRN2 gene, which is a p65 ubiquitin E3 ligase that binds to the p65 subunit and promotes proteasome-dependent degradation, thereby suppressing p65-mediated NFκB transactivation [RNF125, which encodes an E3 ubiquitin ligase that acts as a positive regulator in the T-cell receptor signaling pathway [DOK3, which encodes a novel adapter molecule, DOK3, which is highly expressed in hematopoietic cells, that is involved in the inhibition of immunoreceptor-mediated nuclear factor of activated T-cell (NFAT) activation and cytokine release [NACC2 gene, which is a transcriptional repressor and an important regulator of the p53 pathway [Gene expression analyses of human VL demonstrated upregulation and downregulation of the expression of transcripts related to the TLR4-IFN-I pathway in asymptomatic subjects and VL patients, respectively, and thus provide direct evidence showing that the TLR4-IFN-I pathway is related to the asymptomatic form. Indeed, patients with the severe form of VL develop a clinical condition known as cytokine storm, which is characterized by elevated serum levels of pro- and anti-inflammatory cytokines that return to baseline levels after the chemotherapeutic process ,72,73. Ctivation ; RNF125, pathway ,79; DOK3 release  and that release ; and the pathway . Given tL. infantum infection and in turn avoiding an exacerbated immune response and the consequent immunopathology , 129/SvEv Irf1-/- and 129/SvEv Ifnar-/- mice were obtained from the animal facilities at the School of Medicine of Ribeirão Preto, University of São Paulo (Brazil). The mice were housed in temperature-controlled rooms (22–25°C) at the animal facility of the Department of Biochemistry and Immunology (School of Medicine of Ribeirão Preto) and received water and food ad libitum. All the experiments were conducted in accordance with the National Institutes of Health (NIH) guidelines for the welfare of experimental animals and with approval from the Ethics Committee of the School of Medicine of Ribeirão Preto.Six- to 8-week-old mice were used for the infection experiments. Female C57BL/6 (wild type—WT), C57BL/6 L. infantum (isolate HU-UFS14) was cultured in Schneider medium with 20% heat-inactivated fetal bovine serum (FBS), 5% penicillin and streptomycin  and 2% male human urine. The parasite virulence was maintained by serial passaging in BALB/c mice and by culturing in vitro for no more than five passages. The mice were intravenously infected via the retro-orbital plexus with 107 stationary-phase L. infantum promastigotes in 100 μL of phosphate-buffered saline (PBS). The hepatic and splenic parasite burdens were determined using a quantitative limiting dilution assay.C57BL/6 mice were treated intraperitoneally with 100 μg of α-mouse IFNAR antibody  in PBS 1 day prior to infection and 50 μg of antibody once a week until 6 wpi. A corresponding IgG isotype antibody served as a control (Miltenyi Biotec).6 cells/mL) cultured in RPMI 1640 medium supplemented with 10% FBS were infected with L. infantum promastigotes at a 1:5 ratio (cells:parasites). BMDCs were also treated with LPS  at 200 ng/mL.The femurs of naïve mice were washed with medium, and BMDCs were generated over 6 days using complete RPMI 1640 medium supplemented with 20 ng/mL GM-CSF , as previously described . For the6 cells/mL, seeded into 48-well plates in a total volume of 500 μL of complete RPMI 1640 medium  and stimulated with L. infantum crude antigen (50 μg/mL). The cell culture supernatants were harvested after 72 h of culture at 37°C in 5% CO2.Single-cell suspensions were aseptically prepared from spleen tissue samples, diluted to a concentration of 2 × 10in vitro Th1 cell differentiation, isolated CD4+ cells (MACS Miltenyi Biotec) were stimulated with plate-bound α-CD3 (2 μg/mL) and α-CD28 (1 μg/mL) . The experimental Th1 conditions also included the use of rMuIL-12 (5 ng/mL), IL-2 (25 U/mL), and α-IL-4 antibodies (10 μg/mL) in the presence or absence of rMuIFN-β (1000 U/mL) for 96 h. The cells were labeled with CFSE for the proliferation assay and stained for CD4, T-bet and IFN-γ expression. The supernatants were collected for measurement of the IFN-γ level by enzyme-linked immunosorbent assay (ELISA). All recombinant cytokines were obtained from R&D Systems .For The concentrations of IFN-γ and IL-10 in the supernatants of restimulated splenocytes were determined using commercial ELISA kits. For the detection of IFN-γ in the liver, tissue samples were weighed and titrated in 1 mL of PBS Complete  containing a protease inhibitor cocktail. For IFN-β quantification, the supernatant from stimulated BMDCs was recovered after 24 h of stimulation. The levels of cytokines were determined using commercial ELISA kits for IFN-γ and IL-10 (both obtained from BD Biosciences) and for IFN-β (obtained from PBL Assay Science). Wavelength correction and background signals were subtracted from the absorbance values.For intracellular staining, the cells were cultured with PMA (50 ng/mL) and ionomycin  as well as brefeldin A  for 4 h. The cells were then fixed with 4% paraformaldehyde and permeabilized with 0.5% saponin. Cell acquisition was performed using a FACSort flow cytometer. The data were plotted and analyzed using FlowJo software . The antibodies used are listed in the Supporting Protocol.The protocols used for total RNA isolation and mRNA expression analysis by quantitative real-time PCR (qPCR) are detailed in the Supplemental Methods section. The primers used in qPCR are listed in 7 cells; Miltenyi Biotec, Surrey, U.K.) for 15 min at 4°C. The cells were washed, resuspended at 108 cells/mL, and positively selected on a MACS column (Miltenyi Biotec). The purification of the cells was assessed by FACS using anti-CD4 (RM4-5) and anti-CD62L (MEL-14) antibodies. A total of 5 x 106 CD4+ cells derived from WT or Ifnar-/- mice (C57BL/6 background) were transferred to C57BL/6 Rag1-/- mice in 100 μL of PBS. All the mice were infected with L. infantum 1 day after transfer.Leukocytes were isolated from the spleen and lymph nodes and incubated with anti-CD4 magnetic beads , CAAE 0151.0.107.000–07 and CAAE 0123.0.107.000–11.t test and analysis of variance (ANOVA) were applied to data presenting with normal distribution and variance homogeneity. The means obtained for the different groups were compared by one-way ANOVA followed by the Bonferroni post hoc test. Comparisons between two sample groups were performed using unpaired two-tailed Student’s t test. The data are expressed as the means ± SEMs and are representative of two to four independent experiments. Differences were considered significant at *p < 0.05, **p < 0.01, and ***p < 0.001.The analyses were performed using Prism 5.0 software . Each variable from data collected in the experiments was subjected to analysis of normal distribution and homogeneity of variance. Parametric analyses such as Student’s S1 Fig+ cells, G3) or T cells . (C) The myeloid cell gate (G2) was obtained by first identifying CD11b+ cells (G5) and then (D) identifying dendritic cells  and macrophages .(A) The cells were gated based on their forward scatter height (FSC) and area (SSC) within the areas containing the populations of lymphocytes (G1) and myeloid cells (G2). (B) Lymphocytes (G1) were identified as B cells (CD19(TIF)Click here for additional data file.S2 Fig+ CD11chigh cells (A and B), CD11b+ F4/80+ cells (C and D), CD3+ cells (E and F) and CD19+ cells (G and H) from naïve and L. infantum-infected C57BL/6 WT mice at the indicated time points after infection. The data are expressed as the means ± SEMs (n = 4 mice). The statistical significance was calculated by one-way ANOVA with the Bonferroni post hoc test .Percentage and absolute number of TLR4 expression in the populations of CD11b(TIF)Click here for additional data file.S3 FigCd14-/- mice were i.v. infected with 107L. infantum parasites and euthanized at 6 wpi. (A) Representative histograms of MHCII and CD86 expression in CD11b+CD11chigh cells from the spleen of L. infantum-infected mice. (B and C) Absolute number of MHCII- and CD86-expressing splenic CD11b+CD11chigh cells. (D) Representative dot plots showing the production of IFN-γ by CD4+ T cells from the spleen in response to polyclonal restimulation. (E-F) Graph bars representing the percentage (E) and absolute number (F) of IFN-γ-producing CD4+ T cells in the spleen. (H) Parasite loads in the spleen and liver. The data are expressed as the means ± SEMs (n = 4 mice). The statistical significance was calculated by one-way ANOVA with the Bonferroni post hoc test  or Student’s t test (H).(A-H) WT and (TIF)Click here for additional data file.S4 FigTlr4-/- mice were i.v. infected with 107L. infantum parasites and euthanized at 6 wpi. (A and D) Representative histograms of PD-L1 and CTLA-4 expression in the population of CD11b+CD11chigh cells from the spleen of L. infantum-infected mice. (B and E) Percentage and (C and F) MFI of PD-L1 and CTLA-4 obtained for the population of splenic CD11b+CD11chigh cells from naïve and L. infantum-infected mice. The data are expressed as the means ± SEMs (n = 4–5 mice). The statistical significance was calculated by one-way ANOVA with the Bonferroni post hoc test .WT and (TIF)Click here for additional data file.S5 FigIrf3-/- BMDCs were infected with L. infantum (5 parasites:1 cell) or not infected (medium) for 24 h. Graph bars of the MFI of MHCII (A) and CD86 (B) are shown. (C) Representative dot plots showing the production of IL-12p40 by CD11c+ cells. (D) Graph bars showing the percentage of CD11c+ IL-12p40+ cells. (E) The IL-12p40 levels in culture supernatants were measured by ELISA. The data are expressed as the means ± SEMs (BMDCs were assessed in quadruplicate). The statistical significance was calculated by one-way ANOVA with the Bonferroni post hoc test (*p < 0.05 and ***p < 0.001).WT and (TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S1 Protocol(DOCX)Click here for additional data file.


TMC1 and p.S3417del/p.R1407T in MYO15A in two recessive Chinese Han deaf families. Intrafamilial cosegregation of the mutations with the hearing phenotype was confirmed in both families by the Sanger sequencing. Auditory features of the affected individuals are consistent with that previously reported for recessive mutations in TMC1 and MYO15A. The two novel mutations identified in this study, p.M413T in TMC1 and p.R1407T in MYO15A, are classified as likely pathogenic according to the guidelines of ACMG. Our study expanded the mutation spectrums of TMC1 and MYO15A and illustrated that genotype-phenotype correlation in combination with next-generation sequencing may improve the accuracy for genetic diagnosis of deafness.Genetic hearing loss is a common sensory disorder, and its cause is highly heterogeneous. In this study, by targeted next-generation sequencing of 414 known deafness genes, we identified compound heterozygous mutations p.R34X/p.M413T in  Hair cells (HCs) in the cochlea mainly function in converting the sound mechanical waves into the electric neural signals [Congenital hearing impairment is a common birth defect worldwide, occurring in approximately 1-2 per 1000 infants. With increasing age, the prevalence continues to rise to 2.7 per 1000 before age five and 3.5 per 1000 through adolescence [ signals  which ma signals .TMC1 gene is located on chromosome 9q21 and contains 24 exons that encodes a 760 amino acid membrane protein TMC1 with six transmembrane domains [TMC1 may cause both prelingual profound autosomal recessive deafness DFNB7/11 and postlingual progressive autosomal dominant deafness DFNA36 [TMC1 are reported worldwide [The  domains , 13. TMC domains . TMC1 is domains . It has orldwide , with thorldwide , 16.MYO15A gene is located at chromosome 17p11.2 and contains 66 coding exons, which encodes an unconventional myosin protein Myosin XVA [MYO15A mutations are responsible for congenital deafness DFNB3 in human and cochleovestibular dysfunction in shaker 2 mice which shows abnormally short stereocilia bundles and diminished staircase [The HC stereocilia is critical to maintain the function of HC , 18. Theosin XVA . Myosin osin XVA . Myosin osin XVA . MYO15A taircase , 21. It taircase , 23, witTMC1 and MYO15A as the genetic causes of the hearing loss in those families.In this study, we presented the clinical characterization and genetic analysis of two Chinese Han families affected by ARNSHL. Using targeted next-generation sequencing of 414 known deafness genes, we identified compound heterozygous mutations in This study included two nuclear Chinese Han recessive deaf families: Family 1  and FamiThe genomic DNA of peripheral blood was extracted from all subjects using a Blood DNA kit according to the standard protocol . Targeted next-generation sequencing was performed as previously reported , 25. TheFamily 1 has two affected siblings born to two normal-hearing parents . The proThe proband II-1 of Family 2  was a 10TMC1 (NM_138691) were identified as the only candidate mutations consistent with the recessive inheritance. The Sanger sequencing revealed that the mutations cosegregated with the hearing phenotype in Family 1, as the unaffected parents, were heterozygous carriers of single mutations p.R34X (mother I-1) and p.M413T (father I-2), while the affected siblings both had compound heterozygous mutations (TMC1 were classified as pathogenic (PVS1+PS1+PM2+BS2) and likely pathogenic (PM2+PM3+PP3), respectively.Targeted next-generation sequencing of 414 known deafness genes was performed for the probands Family1-II-1 and Family2-II-1. A total of 9 and 13 candidate variants were identified, respectively Table . In Famiutations . These tMYO15A (NM_016239) were considered the only candidate pathogenic variants consistent with the recessive inheritance. The Sanger sequencing confirmed that the mutations cosegregated with the hearing phenotype in Family 2, as the unaffected parents were heterozygous carriers of single mutations p.R1407T (mother I-1) and p.S3417del (father I-2) (MYO15A were classified as pathogenic (PS1+PM2+PM3+PM4) and likely pathogenic (PM2+PM3+PP3), respectively.In Family 2, compound heterozygous variants c.10245_10247delCTC (p.S3417del) and c.4220G>C (p.R1407T) in her I-2) . These tGJB2 are the most frequent causes [SLC26A4, TMC1, and MYO15A especially in Middle East countries where consanguineous marriage is common [TMC1 and MYO15A were not as extensively reported in the literature.Recessive hearing loss accounts for the majority (80%) of genetic hearing loss . Among mt causes , 34, fols common , 35. On TMC1. The p.R34X mutation is the most common TMC1 mutation in Pakistan [In Family 1, we identified compound heterozygous mutations p.R34X and p.M413T in Pakistan . Using pPakistan . This noPakistan . The p.MMYO15A. The p.S3417del mutation deletes a serine3417 residue at the second FERM domain of Myosin XVA. The FERM domain is a protein-binding domain important in cargo transport and cytoplasmic protein connection to the membrane [MYO15A have been associated with ARSNHL  technology has been proven an effective method for genetic testing of hearing loss in recent years. However, previous studies have showed that NGS in deaf patients, especially the sporadic cases, may detect a significant amount of rare, nonsynonymous variants with unknown functional significance and sometimes even results in false-positive diagnosis . In thisTMC1 and p.S3417del/p.R1407T in MYO15A were identified as the pathogenic causes of ARSNHL in two Chinese Han families. Our results expanded the mutation spectrum of those two genes and showed that NGS in combination with genotype-phenotype correlation may provide a more accurate diagnosis for genetic deafness.Compound heterozygous mutations p.R34X/p.M413T in


Cucumber is one of the most important vegetables in the world. The C2H2 zinc finger protein (C2H2-ZFP) family plays an important role in the growth development and abiotic stress responses of plants. However, there have been no systematic studies on cucumber. In this study, we performed a genome-wide study of C2H2-ZFP genes and analyzed their chromosomal location, gene structure, conservation motif, and transcriptional expression. In total, 101 putative cucumber C2H2-ZFP genes were identified and divided into six groups (I–VI). RNA-seq transcriptome data on different organs revealed temporal and spatial expression specificity of the C2H2-ZFP genes. Expression analysis of sixteen selected C2H2-ZFP genes in response to cold, drought, salt, and abscisic acid (ABA) treatments by real-time quantitative polymerase chain reaction showed that C2H2-ZFP genes may be involved in different signaling pathways. These results provide valuable information for studying the function of cucumber C2H2-ZFP genes in the future. Xenopus oocytes [Transcription factors play an important role in plant growth development and abiotic stress responses. More than 60 transcription factor families have been reported in plants . Among t oocytes . Accordi oocytes . C2H2-ZFEPF1 of Petunia hybrid [Arabidopsis thaliana (Arabidopsis) [Arabidopsis [The first C2H2-ZFP gene found in plants was a hybrid . Subsequidopsis) , rice [9idopsis) , potato idopsis) , and othidopsis) ,13. Studidopsis) , yeast [idopsis) , and Arabidopsis , respectArabidopsis [C2H2-ZFP in eukaryotes generally has a specific conserved sequence consisting of 25–30 amino acids: XXCX (1-5)-CX (12)-HX 3-6)-H . The finger contains two to three β-strands in its N-terminal sequence and one α-helix in the C-terminal . In sili-H . These results should provide valuable information for studying the function of cucumber C2H2-ZFPs in the future.Arabidopsis were obtained from the Arabidopsis Information Resource  [−5). Finally, candidate proteins were confirmed with the SMART (http://smart.embl.de/) [http://pfam.xfam.org/) [Cucumber genome sequences were acquired from the Cucurbit Genomics Database . The C2e . A two  × 10−5) . Second, × 10−5) ) BLASTP mbl.de/)  and Pfamam.org/) .Arabidopsis C2H2-ZFP protein sequences were used for phylogenetic analysis. The MEGAX software was used to construct a phylogenetic tree using the maximum likelihood (ML) method with partial deletion of 1000 bootstraps and a WAG model. The results were formatted for display using Evolview V3 [Information on the distribution of cucumber C2H2-ZFP genes on the chromosomes was obtained using the TBtools software . Cucumber and lview V3 ,41.Gene duplication analysis of Trihelix genes in different species was performed using the Multiple Collinearity Scan software toolkit (MCScanX)  with default parameters . We plotThe gene structure of cucumber C2H2-ZFP genes was identified via TBtools. The online Multiple Expectation Maximization for Motif Elicitation (MEME) program version 5.0.5  was usedTo analyze the expression profiles of cucumber C2H2-ZFP genes in different organs, we retrieved public RNA-seq data (accession number: SRP071224) from the National Center for Biotechnology Information’s Short Read Archive database. Analysis of RNA-Seq data from 23 sampled cucumber tissues was performed based on the regular protocol by Yang et al. . The hea2 and stored at −80 °C for RNA isolation.Cucumbers from the inbred line 9930 were used as plant materials. The experiment was implemented at Shanghai Jiao Tong University . Plants were grown in a growth room at day/night temperatures of 24/18 °C with a 16 h photoperiod. When the cucumber seedlings were at the five-true leaf stage, we conducted the following treatments: 100 mM NaCl, 100 g/L Polyethylene glycol (PEG) 6000, 100 μM ABA, and 4 °C. The NaCl and PEG6000 were added into a nutrient solution and hormones were sprayed onto the leaves. For cold treatment, cucumber seedlings were moved into an illuminated incubator. Leaves from three different plants were harvested at 0, 1, 3, 6, 12, and 24 h after treatment. Each experiment was repeated at least three times. All samples were frozen with liquid Nβ-actin gene (GenBank: AB010922) of cucumber was used as an internal control. Geneious software was used to design primers according to the cDNA sequences . Complementary DNA (cDNA) was synthesized from 1 μg of total RNA using a HiFiScript cDNA Synthesis Kit . Quantitative real-time PCR was carried out using the UltraSYBR Mixture  PCR reactions were prepared and performed according to the manufacturers’ protocol. The equences . PCR-ampt method . Each exArabidopsis C2H2-ZFPs as a query sequence. We found 105 candidate genes, and all putative genes were subsequently verified in the SMART and Pfam databases to confirm the existence of the C2H2-ZFP domains. In total, 101 C2H2-ZFP genes were detected and verified.A two BLAST method was used to identify cucumber C2H2-ZFPs using sequences of The basic information for the 101 C2H2-ZFP genes of cucumber is provided in The 101 C2H2-ZFP genes are distributed across seven cucumber chromosomes, ranging from 9 to 24 genes per chromosome . ChromosArabidopsis , drought (PEG), NaCl, and ABA treatments. As shown in To demonstrate if cucumber C2H2-ZFP genes are involved in stress tolerance or response to hormones, 16 cucumber homologous genes of Csa1G043020, Csa2G033890, Csa3G743410, and Csa4G006510) was inhibited, whereas the transcript level of the other six genes was increased. The six upregulated genes were induced significantly at 24 h after cold treatment. Eight genes were mediated by PEG treatment as follows: seven genes were upregulated, and one gene (Csa3G743410) was downregulated. The same eight genes were upregulated after NaCl treatment. The transcript of Csa3G829160 was strongly upregulated (>20-fold) at 6 h after NaCl treatment. Only five genes were mediated by ABA treatment as follows: four genes were upregulated, and one gene (Csa3G117400) was downregulated.Under cold stress treatment, ten genes were induced. The transcription of four genes (Csa3G697940) responded to all four treatments, and two genes (Csa2G033890 and Csa2G382580) responded to three treatments. The remaining thirteen genes were mediated by one or two treatments. The expression profile indicated that C2H2-ZFP genes may be involved in cucumber responses to abiotic stress, especially to low temperature, high temperature, and NaCl treatment.Among the 16 C2H2-ZFP genes, only one gene (Arabidopsis. The genome of cucumber (genome size of 367 Mb) is larger than the genome of Arabidopsis (genome size of 125 Mb) [Arabidopsis. Whole-genome duplication (WGD) is common in angiosperm plants and produces a tremendous source of raw material for gene genesis. Previous research has revealed three WGD events in Arabidopsis. A paleohexaploidy (γ) event occurred in Arabidopsis after the divergence of monocotyledons and dicotyledons. Subsequently, two WGDs (α and β) occurred in Arabidopsis. The recent WGD events have played an important role in the rapid expansion of gene families [C2H2-ZFPs mainly participate in growth development and abiotic stress responses of plants as thoroughly studied in rice , durum w 125 Mb) , but thefamilies . Howeverfamilies ,49, resuArabidopsis but that more orthologous gene pairs are present in cucumber and melon  gene to be closely associated with Arabidopsis ATZFP6 (AT1G67030). Tu is implicated in cucumber fruit tuberculate development [AtZFP6 plays a key role in regulating trichome development [To analyze the evolutionary relationship of cucumber C2H2-ZFPs, a phylogenetic tree was constructed. In elopment . AtZFP6 Csa5G5773 gene to Arabidopsis  [Generally, C2H2-ZFPs with a similar number of exons and motif composition were classified in the same groups . The dom of 176) , but sim of 176) . Motifs Csa6G303740 had the strongest expression in roots. ZAT11, a homologous gene of Csa6G303740, was highly expressed in roots and particularly in root tips [Csa7G428260 transcript signals were detected only in flesh. MaC2H2-1/2 (two C2H2-ZFPs) is involved in the regulation of fruit ripening in banana via transcriptional repression of ethylene biosynthetic genes [Csa3G199020 showed specific expression in flowers. AtZFP1, an Arabidopsis ortholog of Csa3G199020, is highly expressed in the shoot apex and plays an important role in shoot development [C2H2-ZFP genes play a role in specific tissues at certain stages of plant growth development. Transcriptome data from different tissues and periods were used to analyze the expression profiles of cucumber C2H2-ZFPs. oot tips . Csa7G42Arabidopsis Transgenic ATHB7:: GUS plants showed no or very low GUS activity in all tissues under optimal water conditions. However, the GUS activity was obviously induced in plants subjected to a limited water supply [ATHB12, a paralogous gene of ATHB7, has a similar expression pattern and function [r supply . ATHB12,function . Some geCsa3G697940 was induced by all treatments. Correspondingly, Arabidopsis AtSAP5, a homologous Csa3G697940 gene, also responded to environmental challenges, including salt, osmotic, cold, and ABA stress [Many studies have shown that C2H2-ZFP genes respond to abiotic stress and their expression changes after different time points after stress treatment ,47,56. IA stress . PreviouIn this study, 101 cucumber C2H2-ZFP genes were identified and divided into six groups (I–VI). Chromosomal location revealed that the 101 cucumber C2H2-ZFP genes were distributed in all cucumber chromosomes. C2H2-ZFPs with a similar number of exons and motif composition were classified in the same groups. RNA-seq transcriptome data of different organs revealed temporal and spatial expression specificity of the C2H2-ZFP genes. Expression analysis of sixteen selected C2H2-ZFP genes in response to cold, drought, salt, and ABA treatments by RT-qPCR showed that C2H2-ZFP genes may be involved in different signaling pathways. These results provide valuable information for studying the function of cucumber C2H2-ZFP genes in the future.


Our data demonstrated that these improved BE4max set of plasmids provide desired base substitutions at similar efficiency and without any indels compared to the previously reported BE3 and Target-AID plasmids in zebrafish. Our data also showed that AncBE4max produces fewer incorrect and bystander edits, suggesting that it can be further improved by codon optimization of its components for use in zebrafish. The ease of use and robustness of genome editing by CRISPR/Cas9 has led to successful use of gene knockout zebrafish for disease modeling. However, it still remains a challenge to precisely edit the zebrafish genome to create single-nucleotide substitutions, which account for ~60% of human disease-causing mutations. Recently developed base editing nucleases provide an excellent alternate to CRISPR/Cas9-mediated homology dependent repair for generation of zebrafish with point mutations. A new set of cytosine base editors, termed BE4max and AncBE4max, demonstrated improved base editing efficiency in mammalian cells but have not been evaluated in zebrafish. Therefore, we undertook this study to evaluate their efficiency in converting C:G to T:A base pairs in zebrafish by somatic and germline analysis using highly active sgRNAs to  Genetically engineered animal models play a vital role in the study of genes mutated in human diseases. In general, when mutations in a gene are identified in human patients, animal models are developed to prove disease causation (display of similar phenotype), understand disease mechanism and identify potential treatments. Zebrafish offer several advantages as a vertebrate animal model, such as external fertilization, optically transparent embryos, rapid embryonic development, large clutch size, evolutionarily conserved biological pathways and suitability for high-throughput mutagenesis and chemical screening ,2,3,4. TMore than half of disease-causing mutations in human genetic diseases are caused by single-nucleotide changes that lead to missense, nonsense, stop-loss, or splicing mutations . Except Recently developed programmable base editing technology that combines the targeting specificity of Cas9 with a cytidine or adenine deaminase provides an alternate to HDR for generation of zebrafish with C:G to T:A or A:T to G:C substitutions ,26,27,28Targeted AID-mediated Mutagenesis (TAM) and Target-AID, that used cytidine deaminase AID from human or sea lamprey, respectively, were reported [Komor and colleagues  were thereported ,33. Subsreported ,35,36,37reported ,37,38,39reported ,41. ThesTo date, only proof-of-principle base editing experiments using the earlier versions of CBEs, Target-AID, BE3, and modified BE3 with different Cas9 variants, have been reported in zebrafish ,24,25,28All experiments were performed using the wildtype TAB5 zebrafish strain on the National Human Genome Research Institute Animal Care and Use Committee (ACUC) approved protocol G-05-5. Zebrafish husbandry and breeding were performed as previously described in the Zebrafish book . sgRNA targets were selected based on previously reported successful base editing with BE3 in zebrafish . Their sPlasmids for the following four base editing constructs: pCMV_BE4max (Addgene plasmid # 112093), pCMV_BE4max_P2A_GFP (Addgene plasmid # 112099), pCMV_AncBE4max (Addgene plasmid # 112094), and pCMV_AncBE4max_P2A_GFP (Addgene plasmid # 112100)  were kinFor the evaluation of sgRNA activity, embryos were co-injected into the yolk at the one-cell stage with 50 pg sgRNA and 300 pg spCas9 mRNA. For base editing experiments, embryos were co-injected into the yolk at the one-cell stage with 50 pg sgRNA and 600 pg of one of the base editor mRNAs. All injected embryos were evaluated for toxicity at 24 h post fertilization (hpf).twist2 forward (AGGAGCTGGACAGACAGCAG), twist2 reverse (TCAATGTACCTGGATGCGAG), or ntl forward (CAACAGAAGTGACCACAAGG) and ntl reverse (TTGCTCTTACTGGTGGTAGTGC). M13F and PIGtail adapters were added to the forward and reverse primers, respectively, and a third FAM-M13F primer was used for fluorescent labeling of the PCR product. PCR conditions were as follows: 12 min at 95 °C; 40 cycles 94 °C for 30 s, 57 °C for 30 s, 72 °C for 30 s; 72 °C for 10 min. Fluorescent PCR products were run on a 3730 Genetic Analyzer and CRISPR-STAT analysis was performed by comparing the number of peaks in injected embryos compared to their uninjected controls. PCR amplification for sequencing was performed with the same primers and conditions except for the substitution of ntl forward primer with a different M13F-tailed ntl sequencing forward primer (CAACAGAAGTGACCACAAGG) in order to obtain a high-quality sequence at the target site. PCR products were treated with ExoSAP-IT  followed by Sanger sequencing with M13F primer using the BigDye Terminator v3.1 kit . DNA extraction, fluorescent PCR for CRISPR-STAT and Sanger sequencing were performed as previously described ,44. Briehttp://baseeditr.com) by following the “How to use EditR” instructions. Quad plots were used to validate secondary peaks and editing was considered positive when the trace nucleotide(s) had significance above the p-value cutoff of 0.01. From each injection group, 24–48 embryos were collected individually in a 96-well PCR plate at 48 hpf and processed for DNA extraction, PCR and Sanger sequencing using the M13F primer as described above. Sequence chromatograms were analyzed in Sequencher  to identify embryos showing secondary peaks in the target window. We analyzed all sequence chromatograms that showed secondary peaks in the 5 bp target window using the online EditR tool .twist locus and one was designed to target the ntl locus (twist2-T1 (6/8 embryos) and ntl (7/8 embryos) sgRNAs caused indels detected as multiple peaks, whereas twist2-T2 (0/8 embryos) failed to cause indels at detectable levels in our strain of fish (twist2-T1 (hereafter referred to as twist2) and ntl sgRNAs were used for subsequent base editing experiments. Both target sites contain two Cs in the targeting window that can be converted to T with CBEs  at twist2 in two independent founders in 9% and 3% embryos, respectively (4 → G4) (4 → T4) from founder 2, off-target editing (C11 → T11) was observed outside the target window (4C5) simultaneously in the target window of twist2  and incorrect (G6 → T6) editing at the two Gs in the target window (5 → A5)  and mammalian (mouse) models to study human genetic diseases . Knockoutwist2 (C4C5) and ntl (C5C6). However, with the exception of twist2/AncBE4max, correct editing was observed at only one of the two target nucleotides (C4C5 → T4C5 in twist2/BE4max and C5C6 → C5T6 in ntl with BE4max and AncBE4max). Komor and colleagues [twist2, C4 (context TC) would be favored over C5 (context CC) and matched with our data. For ntl, the context should have favored editing of C6 (context CC) over C5 (context AC), but opposite was observed. Thus, we speculate that factors other than context of the adjacent nucleotide play a role in correct editing of a target nucleotide. As shown in lleagues ,41 showe4 → G4 in twist2 and G6 → T6 in ntl) was observed more frequently with BE4max as compared to AncBE4max. Interestingly, all embryos with editing at ntl by BE4max inherited a combination of correct (G5 → A5) and incorrect conversions (G6 → T6), thus converting GG to TA (or CC to TA on the reverse strand) in the target window. Bystander edits were observed either in combination with the correct edits at the target site, e.g., twist2/BE4max (twist2/AncBE4max (Precision and efficiency of editing are important for widespread adoption of a base editor to generate zebrafish models of human diseases. Successful base editing has been demonstrated in zebrafish using earlier versions of CBEs, such as BE3, BE3-zCas9, BE3-VQRCas9, Target-AID ,24,28. H2/BE4max B or indencBE4max . Thus, fPrevious studies have shown that optimization of genome editing nucleases for their specific use in zebrafish using codon optimization has been effective in improving their efficiencies ,26,53. ZIn conclusion, our study showed that BE4max and AncBE4max can be used to generate heritable point mutations in zebrafish without unintended indels. Overall, AncBE4max showed higher editing efficiency and less incorrect base pair conversions or bystander edits compared to BE4max. Therefore, these CBEs make a valuable addition to the zebrafish toolbox for generation of desired point mutations.


A similar level of heterosis in all F1 individuals is expected as they are genetically identical. However, we found variation in rosette size in individual F1 plants from a cross between C24 and Columbia-0 accessions of Arabidopsis thaliana. Big-sized F1 plants had 26.1% larger leaf area in the first and second leaves than medium-sized F1 plants at 14 days after sowing in spite of the identical genetic background. We identified differentially expressed genes between big- and medium-sized F1 plants by microarray; genes involved in the category of stress response were overrepresented. We made transgenic plants overexpressing 21 genes, which were differentially expressed between the two size classes, and some lines had increased plant size at 14 or 21 days after sowing but not at all time points during development. Change of expression levels in stress-responsive genes among individual F1 plants could generate the variation in plant size of individual F1 plants in A. thaliana.Heterosis or hybrid vigour is a phenomenon in which hybrid progeny exhibit superior yield and biomass to parental lines and has been used to breed F In the process of plant breeding, the phenomenon of heterosis has been exploited in various crops and vegetables because of its effect on yield or stress tolerance [Decreased in DNA methylation 1 (DDM1) lead to lower levels of heterosis, supporting a role for epigenetic contributions to heterosis [Heterosis or hybrid vigour is the superior performance of Folerance ,3, but tolerance ,5,6. Theolerance ,8. MutatArabidopsis thaliana [The concept that the superior performance of hybrid is caused by establishment of more favourable gene expression levels relative to the parental lines has been considered . The trathaliana ,16,17,18thaliana . A. thaliana also shows substantial heterosis in vegetative biomass in particular parental combinations [A. thaliana, heterosis is obvious at early developmental stages in increased cotyledon area at a few days after sowing. Larger cotyledon size generates an increase in photosynthetic capacity, suggesting that this increased photosynthetic capacity in hybrids may cause the maintenance and/or magnification of heterosis at later developmental stages [A. thaliana [In addition to crops and vegetables, inations ,23,24,25inations ,8. In thl stages . A similthaliana . 1 plants using microarrays. A number of genes showed a higher expression level in the big-sized F1 plants than in the medium-sized F1 plants; we examined their effect on plant size by overexpression, focusing on genes categorized into ‘transcription factor’. A number of transgenic plants were larger at 14 or 21 days after sowing, suggesting that these genes play a part in the control of plant size.In this study, we found variation in size among individual plants, which were hybrid between the C24 and Col accessions. To identify the genes regulating the altered plant size in individuals with the same genetic background, we compared the transcriptome profile between big- and medium-sized F1 between C24 and Col had a heterosis phenotype in shoots [1 hybrid between C24 and Col was larger than that of parental lines  varied, and the biggest rosette diameter was 2.7 times larger than smallest  relative to the medium-sized F1 plants  and medium-size fractions in rosette diameter at 10 DAS from 103 F1 plants A, the ro1 plants B, and th1 plants C. The bit 14 DAS D, indica1 plants at 14 DAS using the Affymetrix, Arabidopsis ATH1 Genome Array using total RNAs from shoots of the biggest , the second biggest , and two medium-size fractions  of plants  in expression between B1&B2 and M1&M2 , Col/Ler) and their mid-parent values (MPV) at 15 DAS [er hybrids (p < 10−10). The 61 genes (16.9%) overlapped with upregulated genes in both C24/Col and C24/Ler hybrids (er hybrids (er (22.5%) and Col/Ler (13.8%) hybrids .We compared the lists of non-additively expressed genes in aerial tissues between three Ft 15 DAS  with our hybrids , and the hybrids , while 5 hybrids . Of gene hybrids . The ratddm1C24xddm1Col hybrids or between ColxC24 and ddm1Colxddm1C24 hybrids , ERF6, ERF105, WRKY40, DRE BINDING PROTEIN 1B (DREB1B), Salt-inducible zinc finger 1 (SZF1), and SZF2   using quantitative RT-PCR analysis (qPCR) using another set of big-  and medium-sized  F1 plants A. The re× 10-10) . 2 plants derived from medium- (first) and big- (first) sized F1 hybrid between C24 and Col  and medium  fractions of F2 plants as in F1  cellular component, GO molecular function, and GO biological process . The cat hybrids , similar hybrids . A. thaliana  lines, which have a difference of DNA methylation level with the same genetic background, show higher divergence of flowering time and plant height compared to wild-type Col [1 plants. The other possibility is that epigenetic variation was generated by environmental effects. One candidate is differences of light intensity as in this hybrid, there is increased heterosis under increased light intensity [−2·s−1), but this small difference might result in sizes of F1 plants varying.One of the advantages of Fn easier . This unbreeding , the genal lines ,31, and type Col . This phntensity . In our 1 plants, and approximately 30% of genes showing B1&B2 > M1&M2 expression overlapped with genes upregulated in heterotic F1 plants (C24/Col and C24/Ler) compared with MPV. These genes were categorized into response to wounding, defense response, and response to plant hormones such as ethylene (ET), abscisic acid (ABA), salicylic acid (SA), and jasmonic acid (JA). Transcriptome analysis comparing heterotic F1 and MPV suggested several possibilities to explain the heterosis. (1) Decreased expression levels of defence-responsive genes may play an important role in heterosis by reducing energy cost for defence and releasing resource allocation to plant growth [1 plants. However, sometimes the direction of change of expression levels does not match between big- vs. medium-sized F1 plants and the heterotic F1 vs. MPV; i.e., in big-sized F1 plants, more defence-responsive genes were upregulated, and higher expression levels in genes involved in response to ET or SA stimulus were found. These results suggest that these genes are involved in determining the plant size, and any further increase in plant size of heterotic F1 plants may result from a different expression pattern to non-additive expression.We identified differentially expressed genes between big- and medium-sized Ft growth ,24,33. (t growth . (3) Negt growth . (4) Delt growth . In our 1 [1 compared with ddm1 F1, i.e., an opposite expression pattern. The relationship between increased/decreased expression level and plant size does not necessarily match in the big-size F1 plants and the decreased plant size in ddm1 F1, and changing expression levels of these genes might be involved in the change of plant size.Loss of DDM1 function showed a decreased heterosis level compared with wild-type F1 ,36. The 1-aminocyclopropane-1- carboxylate synthase 6 (ACS6), eliminated heterosis [ETHYLENE RESPONSIVE ELEMENT BINDING FACTOR22 (ERF22), which showed an increased plant size at 21 DAS. Expansin (EXP) and Xyloglucan endotransglucosylases/hydrolase (XTH) are known to be involved in loosening cell wall architecture and cell enlargement [XTH genes has been observed in heterotic hybrids and hybrid mimic lines [β-expansin 1 (EXPB1) induced increased plant size at 14 DAS, while overexpression of EXPB3 did not change the plant size. Overexpressing XTH19 showed variation of plant size, and the average plant size was decreased. XTH19 showed tissue-specific expression, specifically in roots [XTH19 expression in vegetative tissues may be negative for vegetative development. A growth–defence tradeoffs model has been proposed for heterosis or hybrid necrosis phenomena [CARBON/NITROGEN INSENSITIVE 1 (CNI1) or Plant defensin 1.2b (PDF1.2b) led to increased plant size at 21 DAS. CNI1 is important for the carbon/nitrogen response during the early post-germinative growth, and overexpression of CNI1 causes less sensitivity to change in C/N conditions [CNI1 expression was induced by Pseudomonas syringae pv. tomato DC3000 (Pst. DC3000) infection, and overexpression of CNI1 increased resistance to Pst. DC3000 [CNI1 suppressed the senescence phenotype [PDF1.2b encodes plant defensin and is involved in non-host pre-invasive defence response [1 plants [1 plants, showed an increased plant size at 14 or 21 DAS and a large variation of plant size within the lines. However, increased plant size was not observed at all time points. As plant size or heterosis is regulated by tissue- and stage-dependent transcriptional networks, a possible reason is that overexpression of a single gene is not enough to generate the phenotype, and overexpression of multiple genes may be required. Alternatively, changes in gene expression at particular time points may be important for increased plant size.Heterosis has been suggested to affect ET biosynthesis or signal transduction, and overexpression of the ET biosynthesis gene, eterosis . In thisargement . Upregulic lines ,38. In tin roots . Constithenomena ,41. Overnditions . CNI1 ex. DC3000 . In addihenotype . PDF1.2bresponse . Most tr1 plants ,33,46,471 plants of A. thaliana and identified genes that were differentially expressed between big- and medium-sized F1 plants. These differentially expressed genes tended to overlap with non-additively expressed genes in heterotic F1; however, increases and decreases in expression levels in big-sized plants/heterotic F1 did not always match. Non-additively expressed genes showed tissue and stage specificity [1 generation is not a suitable phenotype for F1 hybrid cultivars of crops, but this phenomenon may allow exploration of the factors necessary for maximizing the potential plant size or for stability of heterosis regardless of environmental effects.We found a variation in size in Fcificity ,18,19. W1 between C24 and Col accessions and its F2 population were used for analysis of plant size, microarray, and qPCR. Plants were grown in a controlled environment (22 °C) under fluorescent lights (150–180 μmol·m−2·s−1) and a 16-h/8-h (day/night) photoperiod. Plants were grown in plastic dishes containing Murashige and Skoog (MS) agar medium supplemented with 1.0% sucrose (pH 5.7) and were transferred to soil at 14 DAS.F1 [1 lines, we found a higher variation of rosette diameter at 14 DAS in C24xCol hybrids compared with parental lines  and Col  plants and crossed one combination each. We used the C24 line as female, and multiple flowers were used for making the F1 . Using 1al lines . Further2 seeds were harvested from individual big- and medium-sized F1 plants.Fhttp://rsb.info.nih.gov/ij/). Rosette diameter was measured for evaluation of plant size and equals the maximum diameter of the rosette as measured between the two largest leaves. Rosette diameter depends on leaf blade and petiole length. The first and second leaves at 14 DAS were fixed in a formalin/acetic acid/alcohol solution . The leaf was photographed under a stereoscopic microscope, and sizes were determined with Image-J software. After examination of leaf area, they were cleared in a chloral hydrate/glycerol/water solution , and the samples were photographed under Nomarski optics. The palisade cell number per fixed unit area in the subepidermal layer of the centre of the leaf blade between the midvein and the leaf margin was counted. Three independent experiments were performed for examination of seed size, leaf area, and cell size. Statistical comparisons of seed size, leaf area, and cell size were performed using Student’s t-test (p < 0.05).Dry mature seed was photographed under a stereoscopic microscope, and sizes were determined with Image-J software . From 500 ng total RNA, first-strand cDNA was synthesized using random primers by SuperScript III Reverse Transcriptase (Invitrogen). Prior to qPCR, the specificity of the primer set for each gene was first tested by electrophoresis of PCR-amplified products using EmeraldAmp MAX PCR Master Mix (Takara bio) on 2.0% agarose gel in which single products were observed. Absence of genomic DNA contamination was confirmed by the PCR of no RT control. RT-PCR conditions were 95 °C for 3 min followed by 30 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s. qPCR was performed using a Rotor-Gene 3000 Real-Time Cycler (Qiagen). The cDNA was amplified using Platinum Taq DNA polymerase (Invitrogen). PCR conditions were 95 °C for 2 min followed by 40 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s. Expression levels of genes were calculated relative to Isopentenyl pyrophosphate-dimethylallyl pyrophosphate isomerase 2 (IPP2) genes using the comparative quantification analysis method with Rotor-Gene 6 (Qiagen). Data presented are the average and standard error (s.e.) from two or three biological and three experimental replications. Primer sequences are shown in Total RNA was isolated from aerial tissues at 14 DAS in F1 plants was used for probe synthesis. Biotinylated cRNAs were synthesized using the IVT Labeling Kit (Affymetrix). Hybridization and scanning were performed according to the manufacturer’s instructions. Two independent biological replicates were performed. Data were analyzed following [Arabidopsis ATH1 Genome Array (Affymetrix) was used for transcriptome analysis. Total RNA (100 ng) from aerial tissues at 14 DAS from big- and medium-sized F1 samples from each platform [Analysis for enrichment of gene functional ontology terms was completed using the gene ontology (GO) tool agriGO . The bacplatform . StatistXba I and Sac I or Bam HI and Sac I restriction sites to the 5′- and 3′-ends, and PCR products were cloned into pGEM T-easy vector (Promega). The DNA fragment was then inserted into Xba I and Sac I or Bam HI and Sac I restriction sites of the plant expression vector pBI121 under the control of CaMV35S promoter. The constructs were transformed into Agrobacterium tumefaciens strain EHA105, and transformation of Col accession was carried out by the floral dip procedure [The complete coding sequence (CDS) were amplified by RT-PCR using gene-specific primers designed to add rocedure . Positiv2 lines with a single transgene by segregation analysis using T2 plants with chi-square test. T2 plants were grown in plastic dishes containing MS agar medium supplemented with 1.0% sucrose (pH 5.7). At 14 DAS, they were transferred to soil and grown under long-day conditions (16 h light) at 22 °C. The presence or absence of transgene was examined by PCR using neomycin phosphotransferase II (NPTII) primer set (We selected Timer set .


Scientific Reports 10.1038/s41598-020-69286-3, published online 29 July 2020Correction to: This Article contains an error.As a result of an error during figure assembly, NQU A Windowsill Day 5 image in Fig 2B duplicates NQU Hallway Air Day 8 image in Fig 2A. The correct image for NQU A Windowsill Day 5 is shown below as Figure This change does not affect the conclusions of the Article.


SHP1.A9 allele that contains a copia-like retrotransposon insertion in the promoter region, which silences SHP1.A9 expression epigenetically via DNA methylation.Pod shatter resistance of rapeseed is influenced by a  Brassica napus L.). However, the molecular mechanisms underlying pod shatter resistance are not well understood. Here, we show that the pod shatter resistance at quantitative trait locus qSRI.A9.1 is controlled by one of the B. napus SHATTERPROOF1 homologs, BnSHP1.A9, in a doubled haploid population generated from parents designated R1 and R2 as well as in a diverse panel of oilseed rape. The R1 maternal parental line of the doubled haploid population carried the allele for shattering at qSRI.A9.1, while the R2 parental line carried the allele for shattering resistance. Quantitative RT-PCR showed that BnSHP1.A9 was expressed specifically in flower buds, flowers, and developing siliques in R1, while it was not expressed in any tissue of R2. Transgenic plants constitutively expressing either of the BnSHP1.A9 alleles from the R1 and R2 parental lines showed that both alleles are responsible for pod shattering, via a mechanism that promotes lignification of the enb layer. These findings indicated that the allelic differences in the BnSHP1.A9 gene per se are not the causal factor for quantitative variation in shattering resistance at qSRI.A9.1. Instead, a highly methylated copia-like long terminal repeat retrotransposon insertion (4803 bp) in the promotor region of the R2 allele of BnSHP1.A9 repressed the expression of BnSHP1.A9, and thus contributed to pod shatter resistance. Finally, we showed a copia-like retrotransposon-based marker, BnSHP1.A9R2, can be used for marker-assisted breeding targeting the pod shatter resistance trait in oilseed rape.Seed loss resulting from pod shattering is a major constraint in production of oilseed rape ( Brassica napus L.) is a major source of edible vegetable oil for human consumption and also provides an important energy resource for stock-feed and biodiesel production. Upon maturity, siliques (pods) of oilseed rape open, dehiscing the seed and causing significant yield loss especially when the crop is harvested after it has fully matured . Pod shattering usually accounts for an approximately 10% yield loss on average, but in certain environments it can cause yield loss up to 50%  and SHATTERPROOF2 (SHP2) (INDEHISCENT (IND) (ALCATRAZ (ALC) (FRUITFULL (FUL) in the valves (REPLUMLESS (RPL) in the replum  as oilseed rape, a gene network involved in pod development and dehiscence has been elucidated . For exaAZ (ALC)  have beeSHATTERPROOF paralogs of Arabidopsis (SHP1 and SHP2) are located in the vicinity of the QTL associated with pod shatter resistance on chromosome A9 (designated as BnSHP1.A9 and BnSHP2.A9) in Australian and Chinese oilseed rape populations, including the R1/R2 doubled haploid (DH) population utilized in this study  retrotransposon promoter insertion contributes to pod shatter resistance in oilseed rape by silencing the expression of BnSHP1.A9 epigenetically via DNA methylation.In the present study, we cloned the JXB online) to validate the association between pod shatter resistance index (PSRI) and the BnSHP1.A9 promoter-specific marker. In 2012 and 2013 winter-cropped environments, the DH lines and accessions of a diverse panel of oilseed rape were planted at Yangluo Research Station, Hubei province, China. All DH lines and diverse accessions were planted in a field following a randomized complete block design with two replications. Each plot (2×1 m) contained three rows and each row had 18 plants. All accessions were evaluated for pod shatter resistance using a random impact test as described previously , R2 (pod shatter prone), and 96 DH (R1/R2 DH) lines that showed segregation for pod shatter resistance (BnSHP1.A9 (BnaA09g55330D) from the B. napus cv. Darmor-bzh genome (www.genoscope.cns.fr/brassicanapus) was used as a template to design specific primer pairs (BnSHP1.A9. DNA amplification was performed in a 20 μl reaction comprising 2 μl (10 μM) of forward and reverse primers, 4 μl 5×TransStart®FastPfu Fly buffer, 2 μl 2.5 mM dNTPs, 2 μl 5×PCR stimulant, 0.4 μl 50 mM MgSO4, 1 U TransStart® FastPfu Fly DNA Polymerase , and 20–30 ng template DNA. After initial denaturation of template DNA at 95 oC for 3 min, PCR was carried out following 34 cycles of 20 s at 94 oC, 30 s at about 5 oC lower than melting temperature of the primer pair used, 1 min kb−1 at 72 oC with a final extension of 5 min at 72 oC. The PCR products were separated by electrophoresis in 0.8% low melting agarose gel and slices containing the target bands were purified before cloning into the pEASY-Blunt Zero cloning vector . Transformation was carried out using Trans1-T1 competent Escherichia coli cells with the heat shock method . Four to six positive clones were randomly selected and sequenced with M13 sequencing primers at Shanghai Sangon Biotech Co., Ltd (http://www.sangon.com/). Cloned DNA sequences were analysed by MultAlin online software (BnSHP1.A9 clones by comparison with the Darmor-bzh reference sequence database (http://www.genoscope.cns.fr/brassicanapus/). The upstream sequence of BnSHP1.A9 was cloned using two specific primer pairs  of Darmor-bzh. Based on the sequence differences we developed a co-dominant marker (IF4) that identified an insertion/deletion (InDel) difference in the first intron of BnSHP1.A9 between the parental lines of the R1/R2 population. This marker was used for subsequent genetic mapping and the allelic diversity analysis . The PCR condition used was as follows: 3 min at 94 oC, 35 cycles of 30 s at 94 oC, 30 s at 57 oC, 30 s at 72 oC, with a final extension of 5 min at 72 oC. The PCR products were examined on either 3% agarose gel by electrophoresis at 130 V for 1 h or using capillary electrophoresis on an automated CEQ2000 system (Beckman-Coulter) as described previously . The empirical threshold was computed using 1000 permutations  as described in The 96 DH lines derived from the R1/R2 cross were genotyped with the InDel marker IF4 and other new InDel and simple sequence repeat (SSR) markers developed based on sequence variation between the two parental lines in the vicinity of the 9 region . The InDBnSHP1.A9, tissues from root, stem, and young leaf were collected from 4-week-old plants. Samples from bud, fully open flower, and developing pods were also collected at 10, 20, 30, and 40 d after flowering (DAF) from R1 and R2 parental and DH lines (DH56 and DH82). For each tissue and development stage, three biological replicates were collected with the resulting plant material immediately snap frozen in liquid nitrogen. Total RNA was extracted using the RNAprep Pure Plant Kit . DNAse I-treated RNA was reverse transcribed using the cDNA synthesizing kit following the manufacturer’s instructions . To eliminate interference from other paralogs in the semi-quantitative reverse transcription PCR (RT-PCR), an allele-specific primer pair BnSHP1.A9-RT (F/R) was designed  individually and then used for transformation of the oilseed rape line R1 (resistant to pod shatter)  was extracted from 20 DAF siliques of R1 and R2 and digested with 5 U McrBC overnight at 37 oC. Methylated plasmid DNA was used as positive control in this experiment. The McrBC-digested DNA was then used as a template to amplify the BnSHP1.A9 promoter and genic region with allele-specific primer pairs  to confirm the specificity. The methylation status of the specifically amplified fragments was then analysed by the Kismeth online tool (http://katahdin.mssm.edu/kismeth/revpage.pl). The data for methylated cytosines  between the two parental lines were collected and compared by Student’s t-test.McrBC endonuclease  was used to investigate the DNA methylation status of the er pairs . The ChoBnSHP1.A9 gene from R1 and R2 parental lines of the R1/R2 DH mapping population. The size of BnSHP1.A9 genomic sequences varied from 2737 bp in R1 to 2757 bp in R2, with seven exons and six introns (BnSHP1.A9.Pod shatter tolerance is a complex quantitative trait and is affected by genotype by environment (such as temperature and plant maturity) interaction. In this study, we focused on pod shatter resistance at the full maturity stage when more than 90% of the seed turned black. We isolated the genomic sequences of the  introns . Sequenc introns . HoweverBnSHP1.A9 is associated with pod shatter resistance in a R1/R2 DH population, we developed a primer pair (IF4) for specific amplification of BnSHP1.A9 alleles   . The marectively ; Table 1, BrEMS4 ; BnSHP1.sistance .BnSHP1.A9, we investigated its expression pattern in root, stem, leaf, bud, flower, and developing siliques in R1 and R2, the parental lines of the mapping population. Since there is another homologous copy of SHP1 on chromosome C08 (BnaC08g29520D), to eliminate potential non-specific amplification, we designed a BnSHP1.A9-specific primer pair for RT-PCR based on SNPs between coding sequences of these two homologs. The result showed that BnSHP1.A9 was expressed exclusively in bud, flower, and developing siliques in R1. In contrast, almost no expression in either vegetative organs or reproductive organs could be detected in R2 . A total of 71 transgenic plants were generated and subsequently assessed for the PSRI by random impact test. The PSRI of the transgenic T1 plants varied from 0.35 to 0.53, in comparison with the ‘wild-type’ (untransformed) R1 plants, averaged 0.83 (n=8) (BnSHP1.A9R1 and BnSHP1.A9R2 overexpression (OE) lines. For example, the four OE lines T18, T24, T26, and T33 of BnSHP1.A9R1 had similar PSRI to that of the T9 line of BnSHP1.A9R2  . HoweverHP1.A9R2 .BnSHP1.A9 in developing pods in 15 selected transgenic T1 plants (three from each T1 line) was examined using semi-quantitative RT-PCR. All the tested T1 plants exhibited an elevated level of BnSHP1.A9 expression (BnSHP1.A9 gene (R1 and R2) can promote pod shattering in oilseed rape. The overexpression of BnSHP1.A9 in transgenic plants did not alter flowering time and duration of flowering  (B. rapa A9 scaffold (LR031568.1), which suggested that this LTR retrotransposon insertion may have originated from B. rapa in the R2 paternal line of the R1/R2 DH mapping population.To gain an understanding of the promoter region of the two entation . This LTBnSHP1.A9R2 allele, it may play a role in the repression of the BnSHP1.A9 gene. To further test this hypothesis, we investigated the 96 R1/R2 DH lines using promoter-specific primers of BnSHP1.A9 and selected two DH lines, DH56  and DH82 , which had the same genotype as BnSHP1.A9R1 and BnSHP1.A9R2, respectively. We then collected 10, 20, and 30 DAF siliques and analysed BnSHP1.A9 expression level in siliques of DH56 and DH82 lines by RT-PCR. A weak band was visible in the siliques of all three developmental stages of DH56, which does not contain the LTR insertion (LTR−), while there was no amplification in any of the three silique samples of DH82 containing the LTR insertion (LTR+)  in the DH82 line.As the LTR element is inserted upstream of the start codon of the n (LTR+) . Based oBnSHP1.A9, we performed site-specific chop-PCR and bisulfite sequencing of genomic DNA extracted from 20 DAF siliques of R1 and R2 to analyse the DNA methylation status of the upstream and genomic regions of BnSHP1.A9. Chop-PCR amplified no bands from the LTR insertion fragments  of R2  of R2 , E becau6) of R2 , D. Thesthylated . The metthylated , C. In tBnSHP1.A9 (R1) allele  and homozygous BnSHP1.A9 (R2) allele  were detected in 63.7% (86) and 15.6% (21) of the accessions, respectively, and the heterozygous BnSHP1.A9 (H) allele was found in 11.9% (16) of the accessions  promoter allele and PSRI was observed among the tested oilseed rape accessions  genotype was significantly higher than that of lines with BnSHP1.A9 , indicating that the BnSHP1.A9  allele could increase the PSRI compared with BnSHP1.A9  allele.To verify the linkage between this LTR retrotransposon insertion and pod shatter resistance, we tested 135 diverse accessions with an allele-specific diagnostic marker for LTR  and functional characterization via comparative expression analysis and transgenic approaches, we showed that the SHP1 paralog BnSHP1.A9 underlies pod shatter resistance at qSRI.A9.1 in oilseed rape.In this study, we identified two QTLs, om R1/R2 . The QTLBnSHP1.A9 is the functional gene regulating pod shatter resistance in oilseed rape. By overexpressing BnSHP1.A9 cDNA from both R1 and R2 alleles, an average 50% decrease in PSRI was observed in T1 lines, thus confirming the function of BnSHP1.A9 in pod shatter regulation in oilseed rape  are well known to play positive roles in generating genomic novelty and diversity in plants  is in a transcriptionally active state. DNA methylation of the LTR insertion in the BnSHP1.A9 promoter  spreads to the transcription initiation region, thus converting BnSHP1.A9 from an active state to a silenced state. In this study, hypermethylation of the BnSHP1.A9 promoter region, mainly through CG and CHH methylations, appears to be the major epigenetic factor in the regulation of gene expression. It seems reasonable for plants to evolve such an epigenetic regulatory mechanism to gain function for pod shatter resistance.B. napus germplasm and showed that a single gene, BnSHP1.A9, controls genetic variation for pod shatter resistance at the qSRI.A9.1 locus. Our research provides two gene-specific markers, one co-dominant marker (IF4) detecting a sequence difference within the CDS of BnSHP1.A9, and the other co-dominant marker (BnSHP1.A9p1/p2) detecting presence/absence variation of LTR insertion in the BnSHP1.A9 promoter region. Both markers can be applied for the efficient selection of this QTL for pod shatter resistance in oilseed rape breeding programs starting from early generations. Previously, there were only linked markers reported for marker-assisted selection of pod shatter resistance in oilseed rape (BnSHP1.A9 (R2) allele with positive effect into lines containing the BnSHP1.A9 (R1) allele for varietal improvement. These markers could be easily assayed via conventional agarose gel/high throughput capillary electrophoresis and KASP assays. The sources of pod shatter resistance identified herein can be used for introgression of favorable alleles and enrichment of alleles in the breeding germplasm.In this study we investigated the molecular basis of pod shatter resistance in oilseed rape utilizing natural variation in IND and ALC genes has improved the pod shatter resistance in oilseed rape b). Our results clearly revealed that down-regulation of BnSHP1.A9 could increase the PSRI. Genome editing can not only mutate BnSHP1.A9, but also other functionally redundant homologs of SHP1, as well as the homologs of SHP2. Knockdown of the multiple copies of functionally redundant genes or homologs has more effect on phenotypic variation. However, these approaches are difficult to deploy commercially due to the legal restrictions on genetically modified crops in some European countries. In countries that are open to gene-edited crops, our finding on the down-regulation of BnSHP1.A9 having a positive effect on pod shatter resistance can be used to manipulate gene expression by generating mutation in this gene and its homologs for the improvement of pod shatter resistance in oilseed rape and reduce production losses.Recently CRISPR–Cas9 genome editing and other genetic transformation platforms have become available for oilseed rape improvement  genotypes detected in the natural population.Table S1. The BnSHP1.A9.Table S2. The primers used to amplify genomic sequences, CDS and spatial and temporal expression of Table S3. The primer information of SSR and InDel markers used for genotyping a doubled haploid mapping population derived from the R1/R2 and saturation of marker density of A9 linkage group.Table S4. McrBC digestion chop-PCR primers with gDNA template from R1, R2 20 DAF silique.BnSHP1.A9 promoter.Table S5. The primer information for bisulfite sequencing regions spanning the upstream region of Table S6. The primer information for hygromycin resistance gene used to screen T0 and T1 transgene positive lines.BnSHP1.A9 SNP/Indel variations that were detected between R1 and R2 parental lines.Table S7. The Table S8. The detailed information on the development of the IF4 marker used for genetic analysis of R1/R2 DH population.Table S9. Pod shatter resistance index (PSRI) of transgenic and parental lines of R1/R2 doubled haploid population.eraa281_suppl_Supplementary_Table_S1Click here for additional data file.eraa281_suppl_Supplementary_Table_S2Click here for additional data file.eraa281_suppl_Supplementary_Table_S3Click here for additional data file.eraa281_suppl_Supplementary_Table_S4Click here for additional data file.eraa281_suppl_Supplementary_Table_S5Click here for additional data file.eraa281_suppl_Supplementary_Table_S6Click here for additional data file.eraa281_suppl_Supplementary_Table_S7Click here for additional data file.eraa281_suppl_Supplementary_Table_S8Click here for additional data file.eraa281_suppl_Supplementary_Table_S9Click here for additional data file.eraa281_suppl_Supplementary_FiguresClick here for additional data file.eraa281_suppl_Supplementary_DataClick here for additional data file.


HSD17B14) modulates steroid synthesis and metabolism. To identify the functional single nucleotide polymorphisms (SNPs) in the porcine HSD17B14 gene, ear tissues from Large White and Mi gilts were collected to extract genomic DNA. Variable lengths of truncated promoter of HSD17B14 gene were used to determine the promoter activity by a dual luciferase reporter system. The vector HSD17B14Phe or HSD17B14Val was transfected into porcine granulosa cells (GCs). The core promoter region was identified between −72 bp and −218 bp. Six of seven SNPs had significant differences of allele frequency between Large White and Mi gilts. The plasmids with the wild genotype AA of rs329427898 maintained a smaller fraction of promoter activity compared with the plasmids with the mutant genotype GG, while the plasmids with wild the genotype TT of rs319864566 had a greater promoter activity than the plasmids with the mutant genotype CC. A missense mutation  caused changes in the structural dynamics and function of the HSD17B14 protein. The highly expressed HSD17B14Val degraded less estradiol into estrone, while the relatively lowly expressed HSD17B14Phe degraded more estradiol into estrone, suggesting the protein activity of HSD17B14Phe was greater than that of HSD17B14Val. Moreover, the HSD17B14Phe group has a greater apoptosis rate of porcine GCs. The HSD17B14 gene could been used as a candidate molecular marker for estrus behavior in pigs.Steroid hormone levels are associated with estrous behavior, which affects timely mating and reproductive efficiency in pigs. 17β-hydroxysteroid dehydrogenase type 14 ( The beginning of the estrous cycle occurs at the onset of estrus when the female animal is sexually receptive (known as in heat) and is followed by the ovulation of follicles . In sowsPositive correlations have been found between serum estrogen concentrations and the intensity of estrus behavior of gilts ,8. Repro2), 5-androstene-3β, 17β-diol (5-diol), and testosterone (T), have been identified as substrates of this enzyme [2) into estrone (E1) both in vivo and in vitro [HSD17B14 could allow “control by access” by limiting exogenous and endogenous active estrogen (estradiol) reaching ERs (estrogen receptors) located in the nucleus [As a family of multifunctional enzymes, 17β-Hydroxysteroid dehydrogenases (17β-HSDs) modulate the synthesis and metabolism of steroids by reducing 17-ketosteroids or oxidizing 17β-hydroxysteroid to produce NAD(P)H or NAD(P)+ as a cofactor . 17β-HSDs enzyme ,15. It cin vitro . Moreove nucleus ,17. Estr nucleus ,9.Previous studies have shown that Chinese indigenous pig breeds, such as Meishan, Erhualian, and Mi pig breeds, have superior reproductive performance and estrus expression traits than European pig breeds ,19,20,21HSD17B14 gene was greater in Mi gilts than in Large White gilts [HSD17B14 gene. The objective of this study was to identify the functional single nucleotide polymorphisms (SNPs) of the HSD17B14 gene associated with differences in estrus behavior observed between the Large White and Mi gilts. Several SNPs in the porcine HSD17B14 gene have been identified with significant differences of genotype frequency between Large White and Mi gilts. The effects of these SNPs on the expression of the porcine HSD17B14 gene were investigated in vitro. Furthermore, we verified the effects of a missense mutation in the fifth exon of HSD17B14 on expression, function and apoptosis in porcine ovarian GCs.Our previous study found that the expression level of the te gilts . We hypoThis study was approved by the Animal Care and Use Committee of Nanjing Agricultural University (SYXK2017-0007). A total of 100 gilts (50 Large White and 50 Mi gilts) were randomly selected in Yongkang Agricultural Science and Technology Co., Ltd. in Changzhou, Jiangsu, China. Estrus detection was carefully performed twice daily (at 7:00 and 15:00) at pro-estrus. Expression of estrus was defined as standing reflex, and reddening and swelling of the vulva. Inspections to evaluate standing reflex, and reddening and swelling of the vulva were made on gilts individually by an experienced technician using a standardized routine . A sexua®ACS-4004™) were performed using Lipofectamine 2000 . Cells were plated in 12-well plates. On the following day, the HSD17B14 promoter-luciferase plasmid was co-transfected with plasmid pRL-TK  into the 293T cells. Controls were the pGL3-basic and pGL3-control luciferase reporter gene vector instead of the HSD17B14 promoter luciferase plasmids. After 24 h, cells were harvested with luciferase assay buffer . The cell lysates were assayed for luciferase activity using the Promega Dual Luciferase Assay system. For porcine GC culture, ovaries were obtained from seven-month old unstimulated commercial replacement Large White gilts at a local slaughterhouse. The ovaries were quickly washed twice with 75% ethanol and physiologic saline, and a syringe was used to extract the follicular fluid from a follicle with a diameter of 3–5 mm [2 for 48 h.Transfections in human renal epithelial cell-293T cells  at 260/280 nm. To quantify HSD17B14 messenger RNA (mRNA) levels, total RNA was reverse-transcribed using the HiScript III RT SuperMix for qPCR . RT-qPCR was performed on a QuanuStudio 5 using SYBR Green Master Mix . Relative expression levels were calculated by using the 2−ΔΔCt method [GAPDH. PCR reactions were performed in triplicate and the primers used are shown in Total RNA of ovarian tissue was extracted from six Large White gilts and six Mi gilts using TRIzol  according to the manufacturer’s instructions. The purity of RNA was determined with a NanoPhotometerHSD17B14 gene was predicted by Promoter 2.0 (http://www.cbs.dtu.dk/services/Promoter/) [https://www.fruitfly.org/seq_tools/promoter.html) [http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3) [http://www.urogene.org/methprimer/) [HSD17B14 gene were predicted by Target Scan 7.2 (http://www.targetscan.org/vert_72/) [http://bioinf.cs.ucl.ac.uk/psipred/psiform.html) [The promoter region of the porcine omoter/)  and neurer.html) . Putativ=TF_8.3) ,30. Methprimer/) . MicroRNert_72/) . Proteinrm.html) .HSD17B14 gene. PCR reactions were performed using 1.1 × T3 Super PCR Mix . The amplified PCR products were sequenced, and multiple sequence splices and alignments were performed to analyze the SNPs in Large White and Mi gilts using the software DNAMAN 8.0 (https://www.lynnon.com/index.html) and Chromas .Genomic DNA of 100 gilts (50 Large White and 50 Mi gilts) was extracted from ear tissue by a standard phenol/chloroform method. The specific primers  were desHSD17B14 gene was amplified by PCR using Taq DNA polymerase . Subsequently, plasmids containing variable lengths of truncated the porcine HSD17B14 promoter were individually amplified using different forward primers and a common reverse primer . The amplified fragments were then inserted into the multiple cloning site of the pGL3-basic vector to generate luciferase reporter plasmids. Specific regions containing SNP rs342163057 were amplified using HSD17B14-P5 primers and SNPs rs329427898 and rs319864566 were amplified using HSD17B14-P6 primers. The forward and reverse primers contained Sac I and Hind III recognition sequences.The promoter region of the porcine e primer . For plaHSD17B14Phe and HSD17B14Val overexpression plasmids were built, porcine HSD17B14 CDS (For the missense mutation rs342747498 (T > G), 7B14 CDS  was amplv/v) and total protein extracts were separated on 4–20% SDS–PAGE gels  and blotted onto polyvinylidene fluoride (PVDF). After membranes were blocked with 5% bovine albumin (BSA) in TBST  buffer at 4 °C for 2 h, the membranes were incubated overnight with the following primary antibodies: Immunoreactive proteins were detected with a rabbit polyclonal antibody for HSD17B14  and a rabbit polyclonal antibody for β-actin . The appropriate secondary antibodies were used by anti-rabbit  and chemiluminescence was detected by an Image LAS-4000 system. The band density was analyzed using ImageJ software.Cell protein lysates were collected using 200 μL radio immunoprecipitation assay (RIPA) buffer with 1% Phenylmethylsulfonyl fluoride (PMSF)  and y = 0.0012x + 0.043 (R2 = 0.9942 > 0.95), respectively. The absorbance value  of each well was measured at a 450 nm wavelength in accordance with the operation manual. E1 and E2 levels under different experimental conditions were measured using a Porcine E1 ELISA Kit and an E2 ELISA Kit .The cell culture medium (about 2 mL) was collected 48 h after transfection with pcDNA3.1, GCs were harvested at 48 h after transfection and fluorescence-activated cell sorting (FACS) was performed to measure apoptosis using a cell counting machine . The experiments were conducted using an Annexin V-FITC/PI Apoptosis Detection Kit  according to the manufacturer’s protocol. In total, 10,000 cells were detected and apoptosis rate was calculated. The data were analyzed using the FlowJo v7.6 software .https://www.broadinstitute.org/haploview/downloads). Statistical analyses were performed using IBM SPSS Statistics for Windows . The differences of genotypic frequency between breeds were analyzed using the chi-square test. An unpaired two-sided student’s t-test and one-way analysis of variance were used to evaluate the significance of statistics. Means were reported as means ± standard error of the mean (SEM). Tests were considered significant at p < 0.05.Linkage Disequilibrium (LD) and haplotype distributions of SNPs were analyzed using the expectation maximization algorithm in Haploview 4.2  of gilts in diestrous and estrus (HSD17B14 gene had a greater expression level in the liver than the other seven tissues (p < 0.05) (p = 0.0013), heart (p = 0.0134) and spleen (p = 0.0435) (HSD17B14 gene expression level in the ovary was greater in Mi gilts than in Large White gilts (p = 0.0326) (HSD17B14 was greater in Mi pigs than in Large White pigs (p = 0.0177) (The mRNA expression levels of the d estrus a. The po < 0.05) a. Howeve 0.0435) a. The HS 0.0326) b. At the 0.0177) c.HSD17B14 gene were identified by Promoter 2.0 and neural network promoter prediction. Three CpG island signals  were identified using Meth-Primer 2.0. The 5’-UTR region of porcine HSD17B14 gene promoter harbors potential binding sites for multiple transcription factors including ENKTF-1, C/EBPbeta, NF-1, STAT4, p53, Sp1, FOXP3, AR, E2F-1, Elk-1, NFI/CTF, AP-1, c-Fos (Two promoter regions (−1757/−1668 and −574/−524), and three transcription initiation sites  of the porcine 1, c-Fos .p < 0.05) in allele frequency were found between large white and Mi pigs in six of the seven SNPs (p > 0.05) (p = 1), while the other six SNPs deviated from the Hardy–Weinberg equilibrium (HW p < 0.05) , rs329427898 (−683A > G), and rs319864566 (−502T > C), were identified in the 5’-UTR; three SNPs, rs329068902 (+556C > A), rs318859497 (+573G > A), and rs337682650 (+574T > G), were identified in the first intron, and rs342747498 (+2096T > G) was identified in the fifth exon a. Signifven SNPs . The num > 0.05) . In Mi p < 0.05) . Linkage < 0.05) b; while  < 0.05) c.HSD17B14 gene was isolated to determine the SNPs that are responsible for influencing the function of the promoter. The luciferase reporter plasmids, named PGL3-basic-P1 (−2057/+254), PGL3-basic-P2 (−1273/+254), PGL3-basic-P3 (−972/+254), PGL3-basic-P4 (−218/+254), were transfected into 293T cells. The luciferase activity of plasmids with one of four HSD17B14 promoter fragments was greater than that of the pGL3-basic group (p < 0.01) and less than that of the pGL3-control group (p < 0.01). The luciferase activity of PGL3-basic-P3 was greater than that of PGL3-basic-P1 (p = 0.0402) or PGL3-basic-P2 (p = 0.0496) (p < 0.05). These results show that the core promoter region of the HSD17B14 gene is located between −972 bp and −218 bp, whereas a negative regulatory promoter region is located between −1273 bp and −972 bp.The proximal minimal promoter of the porcine  0.0496) . MoreoveHSD17B14 gene, rs342163057 (−850G > C) and rs329427898 (−683A > G) are located at downstream of the promoter region and rs319864566 (−502T > C) is located at the core promoter region. Promoter activity of different genotypes of rs342163057 (−850G > C), rs329427898 (−683A > G), and rs319864566 (−502T > C) in the porcine HSD17B14 gene were analyzed using a dual-luciferase reporter assay system. As shown in p < 0.05). In p < 0.01) but less than those of the pGL3-control group (p < 0.01). Luciferase reporter plasmids with combined genotype AATT had less (p = 0.0451) luciferase activity than that of plasmids with combined genotype GGTT. In addition, the plasmids with combined genotype AACC had lower (p = 0.026) luciferase activities than those of plasmids with combined genotype GGCC. These results suggest that the plasmids with genotype AA had lower luciferase activities than those of plasmids with genotype GG of rs329427898 (−683A > G). At the same time, the plasmids with combined genotype GGCC have lower (p = 0.0007) luciferase activities than those of plasmids with GGTT, while the plasmids with combined genotype AACC had lower (p = 0.0001) luciferase activity than that of plasmids with combined genotype AATT of rs329427898 (−683A > G) and rs319864566 (−502T > C). These results suggest that the plasmids with genotype CC had lower luciferase activities than those of plasmids with genotype TT of rs319864566 (−502T > C).In the seven SNPs of the HSD17B14 gene, according to the information in Ensembl (https://www.ensembl.org/index.html). It causes a mutation from phenylalanine (F) to valine (V) at the seventy-third amino acid of the HSD17B14 protein. Since the genotype of rs342747498 was TT in Large White pigs and GG in Mi pigs, the protein sequences of HSD17B14 were totally different between the Large White (HSD17B14Phe) and Mi  pigs. Predicted protein structure figures show that the three-dimensional structures of the HSD17B14 protein are different between Large White and Mi pigs (The SNP rs342747498 (T/G) is located in exon five of the porcine  Mi pigs a,b. The  Mi pigs a,b.HSD17B14Phe or HSD17B14Val were transfected into porcine GCs. Subsequently, the mRNA expression level of HSD17B14Val was significantly greater than that of HSD17B14Phe (p < 0.05), whereas the mRNA expression level of HSD17B14Val or HSD17B14Phe was greater p < 0.05) than that of pcDNA3.1 , while the protein expression levels of both HSD17B14Val and HSD17B14Phe were greater (p < 0.05) than those of pcDNA3.1  than those of GCs in the HSD17B14Phe group  than those of GCs in the HSD17B14Phe group (Vectors pcDNA3.1 (empty vector), pcDNA3.1 c. The aspcDNA3.1 d,e. In ahe group d, while he group e.HSD17B14Val and HSD17B14Phe proteins, a HSD17B14Phe or HSD17B14Val overexpression vector was transfected into porcine GCs. RT-qPCR of BAX and BCL-2 genes showed that the ratio of BCL-2/BAX was significantly less (p < 0.05) in the HSD17B14Phe group than in the HSD17B14Val group, while it was less (p < 0.05) in the HSD17B14Phe or HSD17B14Val group than in the pcDNA3.1 group  than that caused by HSD17B14Phe. A fluorescence activated cell sorting (FACS) analysis also revealed that the GCs in the HSD17B14Phe group had a greater (p < 0.05) apoptosis rate than the GCs in the HSD17B14Val group , into estrone (E1) both in vivo and in vitro [HSD17B14 gene was differentially expressed during the diestrous and estrus periods between Large White and Mi gilts [HSD17B14 gene could affect the estrus of gilts by regulating the transformation of estradiol to estrone.Gene expression analyses and tissue-specific expression profiling might be useful for functional research and gene mapping. ,36. EstrMi gilts . Thus, tHSD17B14 gene was identified and located at a CpG island (−667 bp and −562 bp)  , which ipression . Moreovepression , for proHSD17B14 gene was significantly different between the two stages of the estrus cycle and between the two pig breeds [HSD17B14 gene. Three SNPs in the promoter region caused the different expression level of the porcine HSD17B14 gene between Mi and Large White gilts. Two SNPs in the first intron acted on the transcription of the HSD17B14 gene through linkage with the SNPs in the promoter region in Large White gilts. However, rs318859497 was also linked with the SNPs in the promoter region, in addition to rs329068902 and rs337682650 in Mi pigs. In addition, as the result of a missense mutation, an encoded amino acid changed from phenylalanine (F) to valine (V) in the fifth exon. Previous studies had shown that certain specific missense mutations not only affected the function of the target protein and the incidence of disease [Our previous studies found that Chinese Mi gilts had more intense estrus behaviors and greater estradiol-17β concentrations than Large White gilts . The expg breeds . In the  disease , but als disease . For exa disease .HSD17B14 gene were not located in the CpG island. Because the luciferase activities of plasmids with GG and CC genotypes were less than those of the negative control—the pGL3-basic group (p < 0.05)—there was a transcription-inhibiting element that binds to CC and GG genotypes (the binding energy of the CC genotype was greater than that of GG), resulting in the luciferase activity of rs342163057 (−850G > C) being less than that of PGL3-basic. For the other two SNPs rs329427898 (−683A > G) and rs319864566 (−502T > C), the combined genotypes  showed a progressive increase and significant differences between them. The luciferase activities of plasmids with GG and TT genotypes were greater than those of plasmids with AA and CC genotypes, which suggests that these two SNPs can affect the promoter activity of the HSD17B14 gene. In addition, rs342163057 (−850G > C), rs329427898 (−683A > G) and rs319864566 (−502T > C) were located in the positive regulatory region in the promoter. Compared to rs329427898 (−683A > G), rs319864566 (−502T > C) is the dominant SNP for regulating the porcine HSD17B14 gene.Polymorphisms in the 5’-UTR of a gene could affect its transcription . In the HSD17B14 gene. They were reported to have important influences in tumorigenesis and cell growth [In the present study, we predicted several transcription factors, such as STAT4, TFII-1, c-Ets-1, and NF-AT2  for the l growth .HSD17B14 gene, the catalytic activity of the enzyme had a larger change than before the mutation [HSD17B14 gene in the fifth exon in Large White and Mi gilts. The missense mutation could lead to changes in the structural dynamics, which are determinant of the functional significance of missense variants [HSD17B14 protein, including strand and helix structure. These changes could affect the enzyme activity of the HSD17B14 protein [A missense mutation is an inheritable alteration in the sequence of the genetic material of an organism . In the mutation . In the variants . Protein protein , which mHSD17B14 were significantly lower in the overexpression vector HSD17B14Phe group than in the HSD17B14Val group in the present study. These results indicate that more highly expressed HSD17B14Val did not degrade more estradiol into estrone, while the relatively lesser expressed HSD17B14Phe degraded more estradiol into estrone, suggesting the protein activity of HSD17B14Phe was greater than that of HSD17B14Val. In this situation, the low protein activity of HSD17B14Val did not cause a significant reduction in estradiol in porcine ovarian GCs, and the feedback regulation would increase the mRNA and protein levels of HSD17B14Val. These results further illustrate why the expression level was greater for the HSD17B14 gene in Mi gilts than in Large White gilts. The results also explained why Chinese Mi gilts had better estrus behavior traits than Large White gilts.In addition, both mRNA and protein levels of 2 promoted the proliferation of porcine GCs in a time- and dose-dependent way [2 abolished gonadotropin-mediated pro-apoptotic signals in human primary granulosa lutein cells [2 also prevented H2O2-induced apoptosis of large luteal and granulosa cells of pigs [HSD17B14Phe degraded more estrogen than HSD17B14Val. Thus, apoptosis of pig ovarian granulocytes was greater in HSD17B14Phe than in HSD17B14Val. Follicle development is closely associated with the development of GCs [HSD17B14Phe could have a negative impact on ovarian development and the reproductive performance of female pigs, which could inhibit estrus signs of gilts.Estrogen is necessary for the proliferation and apoptosis of porcine GCs ,49. In adent way . Similarin cells . In addi of pigs . In the t of GCs . TherefoHSD17B14 gene in the follicles was greater in Mi gilts than in Large White gilts during diestrous, but was lower in Mi gilts than in Large White gilts during estrus [HSD17B14 protein was mainly involved in the transformation from high-efficient E2 to low-efficient E1 in the estrogen metabolism pathway [HSD17B14 gene could affect the binding of RNA polymerase and transcription factors to the promoter region, influencing its expression and the ability to degrade estradiol to estrone. In addition, although Mi gilts showed an increase in protein and mRNA expression levels, the missense mutation of Phe73Val in the fifth exon weakens the ability of the protein to degrade estradiol to estrone, which would make the estrus signs more obvious and longer lasting in Mi gilts  that can be used to screen gilts with better estrus sign traits. Better estrus traits could also improve the accuracy of estrus detection, which increases the efficiency of pig reproduction.Our previous study found that the expression level of the g estrus . The por pathway . TherefoMi gilts . FurtherHSD17B14 gene is located between −972 bp and −218 bp from the initiation site of transcription. A negative regulatory promoter region was found between −1273 bp and −972 bp. The luciferase reporter plasmids with the GG genotype of rs329427898 had greater promoter activities than plasmids with the AA genotype; plasmids with the TT genotype of rs319864566 had greater promoter activities than plasmids with the CC genotype, which is consistent with the estrus difference between Large White and Mi gilts in vivo. In addition, rs342747498 located in exon five of the HSD17B14 gene caused a missense mutation, which lead to phenylalanine (F) being changed to valine (V) at seventy-third amino acid position in the HSD17B14 protein. This had a profound effect on the mRNA and protein expression levels of the porcine HSD17B14. These results indicate that these SNPs in the HSD17B14 gene could be candidate molecular markers for estrus signs in pigs.The promoter activity analyses suggest that the core promoter region of the porcine