牛卵泡发育相关下调基因的筛选  

Screening of down-regulated genes related to follicle development in bovine

作　　者：赵园园 赵成刚[1] 安清明 武渊勋 孟金柱 ZHAO Yuan-yuan;ZHAO Cheng-gang;AN Qing-ming;WU Yuan-xun;MENG Jin-zhu(Tongren University,Tongren,Guizhou 554300,China;Shanxi Pu Yuan Tai Dairy Cattle Breeding Co.LTD,Jinzhojig,Shanxi 030800,China)

机构地区：[1]铜仁学院,贵州铜仁554300 [2]山西普源泰奶牛养殖有限公司,山西晋中030800

出　　处：《中国兽医学报》2020年第9期1825-1831,共7页Chinese Journal of Veterinary Science

基　　金：贵州省教育厅青年科技人才成长基金资助项目(黔教合KY字[2018]348);铜仁市科技计划资助项目(铜市科研[2017]39号);铜仁市科技计划资助项目(铜市科研[2016]18号-1);铜仁学院博士启动基金资助项目(trxyDH1601)。

摘　　要：为寻找牛卵泡颗粒细胞(GCs)凋亡的关键调控因子,阐明单胎动物卵泡闭锁调控机理,本研究对奶牛优势卵泡与从属卵泡GCs深度测序,筛选卵泡发育相关的下调基因。选取健康荷斯坦奶牛,屠宰后,分别采集卵巢上正常发育的最大卵泡(8~10 mm)与第二大卵泡(5~8mm),并结合雌激素/孕激素确定卵泡优势与否,优势卵泡(DF):E2/P>1;从属卵泡(SF)E2/P<1。分别刮取GCs,提取总RNA并建库,Illumina测序,将获得序列与牛Refseq数据库比对后,利用DESeq2软件对获得的RNA进行差异表达分析,并对其中表达下调的基因进行GO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。测序共获得32346个基因,其中194个在DF中表达显著上调,502个表达下调;GO分析结果显示,这些下调基因的生物学功能共分为3大类104组,其中生物学过程相关基因占61.5%,细胞组分有关基因占25.0%,分子功能相关基因占13.5%;KEGG信号通路分析,共发现5条通路,其中基因富集最为显著的是癌症通路;从下调基因中筛选出4个在牛卵泡发育过程中可能会起抑制作用的基因,QRT-PCR结果显示,PPP1R14A、QRFPR和EGR1在DF和SF中的表达趋势与深度测序结果一致,OLA1在DF和SF中的表达趋势与深度测序结果正好相反,但差异不显著。本研究筛选出的4个表达下调基因可能是牛卵泡发育过程中抑制卵泡发育的候选基因。In order to find the key regulatory factors of apoptosis of bovine follicular granulosa cells and clarify the regulatory mechanism of follicular atresia in single-foetal animals,in this study,dominant follicular and subordinate follicular granulosa cells in dairy cows were conducted in-depth sequencing and down-regulated genes related to follicular development was screened.Healthy dairy cows(2 years old)were selected.After slaughter,the largest follicles(8-10 mm)and the second largest follicles(5-8 mm)on the ovaries were collected,respectively.Meanwhile,the value of estrogen/progestogen determines the follicular advanced or not(dominant follicle:E2/P>1,subordinate follicular:E2/P<1).Granulosa cells were scraped and total RNAs were extracted,constructed libraries and sequenced using Illumina.Then database of sequences were matched with the Refseq database of cattle,differentially expressed genes were analyzed by DESeq2 software and GO,KEGG signal pathway analysis of down-regulated genes were executed.Finally the expression levels of down-regulated genes were validated through the real-time PCR.A total of 32346 genes were acquired,among which 194 genes were significantly up-regulated in DF and 502 genes were down-regulated.GO analysis showed that the biological functions of these down-regulated genes were divided into three categories and 104 groups,in which biological process accounted for61.5%,Cellular component related genes accounted for 25.0%,and molecular function related genes accounted for 13.5%.Five pathways were found by KEGG signal pathway analysis,among which the most significant gene enrichment was the cancer pathway.Four genes that might inhibit the development of bovine follicles were selected from the down-regulated genes.QRT-PCR results showed that the expression trend of PPP1 R14 A,QRFPR and EGR1 in DF and SF were consistent with deep sequencing,while the expression trend of OLA1 was opposite to deep sequencing,but the difference was not significant.Four down-regulated genes selected in this study

关 键 词：牛 卵泡发育 下调基因 深度测序 

分 类 号：S823[农业科学—畜牧学]