西藏绒山羊羊绒细度候选基因筛选及其chi-miR-105a靶基因的验证

Screening of candidate genes related to Tibetan cashmere fineness and verification of the relationship between chi-miR-105a target genes

为研究西藏绒山羊羊绒细度,以及了解相关候选基因对绒山羊产绒性能的影响,本研究对筛选chi-miR-105a靶基因进行验证,以西藏绒山羊为研究对象,联合RNA测序数据和蛋白质组学数据,筛选与羊绒细度相关的关键候选基因,并通过实时荧光定量和双荧光素酶报告基因试验对候选基因进行调控作用验证。结果表明:1)通过转录组和蛋白组数据整理得到384个DE mRNAs,12个DE miRNAs和29个DEPs。通过多组学联合分析发现CXCL10、SRC、CXCL9、FOS、ETV6、GMPS和PIP5K1B基因与羊毛细度相关。2)通过DEG-DE miRNA互作网络图发现chi-miR-105a与靶基因ETV6和PIP5K1B均在网络当中出现。因此对该调控因子进行验证和分析,根据靶基因表达水平试验发现转染chi-miR-105a mimics后引起毛乳头细胞中ETV6基因的mRNA表达显著降低(P<0.001),而PIP5K1B基因的表达量均无显著变化。3)通过双荧光素酶报告基因试验表明,过表达chi-miR-105a后,ETV6酶活性显著降低,即chi-miR-105a可与ETV6的3′UTR区结合。综上,通过多组学联合分析筛选出与羊毛细度相关基因CXCL10、SRC、CXCL9、FOS、ETV6、GMPS和PIP5K1B。通过双荧光素酶报告基因进行chi-miR-105a与其预测靶基因ETV6和PIP5K1B的靶标验证,确定chi-miR-105a是ETV6潜在的调控因子,本研究为加快优质绒山羊新品种(系)的培养提供理论依据。

In order to study the cashmere fineness of Tibetan cashmere goats and understand the effects of related candidate genes on cashmere production performance of cashmere goats,this study verified the screening of chi-miR-105a target genes.Taking Tibetan cashmere goats as the research object,RNA sequencing data and proteomics data were combined to screen key candidate genes related to cashmere fineness,and the regulatory effects of candidate genes were verified by real-time fluorescence quantification and dual luciferase reporter gene assay.The results showed that:1)384 DE mRNAs,12 DE miRNAs and 29 DEPs were obtained from transcriptome and proteome data.Through multi-omics joint analysis,it was found that CXCL10,SRC,CXCL9,FOS,ETV6,GMPS,PIP5K1B genes were associated with wool fineness.2)Through the DEG-DE miRNA interaction network diagram,it was found that chi-miR-105a and target genes ETV6 and PIP5K1B appeared in the network.Therefore,the regulatory factor was verified and analyzed.According to the target gene expression level test,it was found that the mRNA expression of ETV6 gene in dermal papilla cells was significantly decreased after transfection of chi-miR-105a mimics(P<0.001),while the expression of PIP5K1B gene was not significantly changed.3)Double luciferase reporter gene assay showed that ETV6 enzyme activity was significantly decreased after overexpression of chi-miR-105a,that is,chi-miR-105a could bind to the 3′UTR region of ETV6.In summary,CXCL10,SRC,CXCL9,FOS,ETV6,GMPS and PIP5K1B related to wool fineness were screened by multi-omics combined analysis.The target of chi-miR-105a and its predicted target genes ETV6 and PIP5K1B was verified by dual luciferase reporter gene,and chi-miR-105a was identified as a potential regulator of ETV6.This study provided a theoretical basis for accelerating the cultwation of new varieties(lines)of high-quality cashmere goats.