中国美利奴羊不同时期卵巢组织cDNA消减文库(SSH)的构建及序列分析  

cDNA Suppression Subtractive Hybridization(SSH) Library Construction and Sequence Analysis of the Ovary of Chinese Merino Sheep(Ovis aries) in Different Period

作　　者：林杉[1] 陈磊[2] 张文祥[2] 李大全[2] 赵宗胜[2] 

机构地区：[1]新疆石河子大学生命科学学院,石河子832003 [2]新疆石河子大学动物科技学院,石河子832003

出　　处：《农业生物技术学报》2014年第5期642-650,共9页Journal of Agricultural Biotechnology

基　　金：国际科技合作项目(No.2007DFB30420)

摘　　要：季节性发情是制约我国绵羊(Ovis aries)产业发展的重要因素之一。母羊卵巢是重要的生殖器官,卵巢组织中周期性特异表达的基因很有可能直接或间接影响母羊的季节性发情。为探讨绵羊季节性发情的分子遗传机制,本研究采用抑制性消减杂交技术构建了发情期发情和未发情中国美利奴羊卵巢组织差异表达的消减cDNA文库。随机取样386个阳性克隆,经PCR鉴定获得有效克隆306个,测序后获得126条非冗余序列;用BLAST在线序列软件与GenBank、GenBank EST等数据库进行同源性序列比较,发现其中有21个未知序列,可能是和中国美利奴羊季节性产羔性状相关的新基因。从文库中选择差异表达的视黄酸受体应答1基因(RARRES1)、胸腺素β4基因(TMSB4X)、翻译延长因子基因(EEF1A1)、前胸腺素α基因(PTMA)以及12号和89号基因,在心脏、肝脏、脾脏、肺脏、肾脏、卵巢、输卵管、下丘脑和垂体中对上述6种基因进行组织表达谱分析。结果发现,TMSB4X、PTMA、EEF1A1和12号基因在上述组织中均有表达,而RARRES1和89号基因仅在中国美利奴羊卵巢组织中高表达。为了进一步验证研究结果,对RARRES1、TMSB4X、EEF1A1、PTMA、12号和89号基因进行实时定量PCR检测,结果显示,上述基因在发情中国美利奴羊卵巢组织中的表达量显著高于未发情绵羊。本研究筛选出调控中国美利奴羊季节性繁殖相关的候选基因,为进一步研究卵巢功能与绵羊季节性繁殖相互作用的分子机制提供了基础资料。Seasonal oestrus is one of main restricted factors in the development of sheep industry. The ovaries are the important reproductive organs of sheep, and the periodicly expressed gene in ovary is likely to regulate the ewe seasonal breeding directly or undirectly. In order to discuss the molecular genetic mechanism of the sheep seasonal oestrus, the subtracted cDNA library of the Chinese Merino sheep（Ovis aries） ovary（estrus and anestrus） was conducted by the suppression subtractive hybridization（SSH）. Random sampling of 386 positive clones were identified by PCR, and obtained about 306 effective clones and 126 non-redundant sequences after sequencing. The sequences were studied through BLAST on line with the GenBank dbase and GenBank EST dbase and the results showed that 21 of them were not matched to any of the databases which might represent new genes related to Chinese Merino sheep ovary tissue seasonal reproduction trait. Retinoic acid receptor response element1（Rarres）, thymosin beta 4（TMSB4X）, eukaryotic translation elongation factor 1 alpha 1（EEF1A1）, prothymosin alpha（PTMA）, and No.12 and No.89 genes were selected from the library, and tissue-specific detection of the 6 kind of genes screened was performed in heart, liver, spleen, lung, kidney, ovary, hypothalamus, pituitary, uterus and fallopian tubes tissues. The results showed that RARRES1 and No.89 gene over-expressed in ovary tissues, however, TMSB4X, EEF1A1, PTMA and No.12 gene expressed in all tissue. In order to further validate the obtained results, RARRES1, TMSB4X, EEF1A1, PTMA, No.12 and No.89 genes were selected for expression analysis by quantitative Real-time PCR. The results showd that their expression were all up-regulated in ovary of estrous Chinese Merino sheep. In this study, we got potential genes related with the seasonal reproduction trait of the Chinese Merino sheep, wihch provides a foundation to research the molecular mechanism of interaction between ovary function and seasonal reproduction trait of she

关 键 词：中国美利奴羊 抑制消减杂交 发情 卵巢 CDNA文库 

分 类 号：S794.1[农业科学—林木遗传育种]