基于RNA-seq技术筛选山羊卵泡发育相关下调基因

Screening down-regulated genes related to follicular development of goats based on RNA-seq technology

【目的】对山羊优势卵泡(DF)和从属卵泡(SF)颗粒细胞进行高通量测序,筛选并研究与山羊卵泡发育相关的下调基因。【方法】以1岁龄贵州白山羊为供试对象,在其发情3 d后,采集第一卵泡波中的优势卵泡(DF)和从属卵泡(SF),分别分离其颗粒细胞,提取RNA并进行文库构建、Illumina测序,将获得的序列与山羊的RefSeq数据库进行比对后,用DESeq2软件对获得的RNA进行差异表达分析,并对表达下调的基因进行GO分析和KEGG信号通路分析,最后通过Real-time PCR对筛选出的表达下调基因进行验证。【结果】将测序结果与山羊的RefSeq数据库比对后,共获得了33896个基因。经DESeq2软件对获得的mRNA进行差异表达分析,共获得695个差异表达m-RNA,其中462个表达显著下调,233个表达则显著上调;对462个表达下调基因进行GO分析,共分为2大类39组,其中参与生物学过程(BP)的基因占48.7%;与细胞组分(CC)有关的基因占51.3%。KEGG信号通路分析发现20条通路,其中参与癌症通路基因富集最为显著。从下调基因中筛选出6个可能会抑制山羊卵泡发育的基因,分别为PTX3、LDLR、RGN、NID1、PDLIM5、PRLR。Real-time PCR结果显示,RGN、NID1、PDLIM5、PRLR在SF与DF中的表达趋势与高通量测序结果一致,且RGN在SF颗粒细胞中的表达量极显著高于DF(P<0.01),NID1、PDLIM5在SF颗粒细胞中的表达量显著高于DF(P<0.05);PTX3、LDLR在SF与DF中表达量与高通量测序结果相反。【结论】获得的6个表达下调基因在山羊卵泡发育过程中可能会抑制卵泡的发育,最终导致卵泡闭锁。

【Objective】This study screened and analyzed the down-regulated genes related to follicular development through high-throughput sequencing for granulosa cells in dominant follicle(DF)and subordinate follicle(SF)of goats.【Method】The DF and SF in the first follicular wave of Guizhou white goats at the age of 1 year were collected 3d after estrus,granulosa cells were isolated,RNA was extracted,libraries were constructed and Illumina sequencing was performed.After comparing the obtained sequences with the RefSeq database of goats,DESeq2 software was used to analyze the differential expression of the obtained RNA,the down-regulated genes were analyzed by GO and KEGG signaling pathway analysis,and the real-time PCR was used to verify the selected down-regulated genes.【Result】After comparing the sequencing results with RefSeq database,a total of 33896 genes were obtained.A total of 695 differentially expressed mRNAs were obtained by DESeq2 software,among which 462 were significantly down-regulated and 233 were significantly up-regulated.The GO analysis divided down-regulated genes into 39 groups of two major categories.The biological process(BP)accounted for 48.7%and cell component(CC)accounted for 51.3%.KEGG signaling pathway analysis revealed 20 pathways with the most significant in enrichment of genes involved in cancer pathways.Six genes of PTX3,LDLR,RGN,NID1,PDLIM5 and PRLR that may inhibit follicular development were selected from the down-regulated genes.Real-time PCR results showed that the expression of RGN,NID1,PDLIM5 and PRLR was consistent with high-throughput sequencing results in SF and DF,and the expression level of RGN in SF granulosa cells was extremely significantly higher than that in DF(P<0.01).The expression levels of NID1 and PDLIM5 in SF granulosa cells were significantly higher than those in DF(P<0.05).The expression levels of PTX3 and LDLR in SF and DF were opposite to the high-throughput sequencing results.【Conclusion】It is speculated that the 6 down-regulated genes obtained in this study may inhibit the development of follicles during follicular development in goats and eventually lead to follicular atresia.