山羊卵巢颗粒细胞差异表达基因消减文库的建立  

Construction of a subtractive library of genes differentially expressed in follicular granulosa cells of goat ovary

作　　者：庞训胜[1,2] 王子玉[1] 应诗家[1] 张艳丽[1] 闫益波[1] 吴勇聪[1] 孟立[1] 王锋[1] 

机构地区：[1]南京农业大学羊业科学研究所,江苏南京210095 [2]安徽科技学院动物科学学院,安徽凤阳233100

出　　处：《南京农业大学学报》2010年第6期95-100,共6页Journal of Nanjing Agricultural University

基　　金：国家科技支撑计划项目(2008BADB2B04;2008BADB2B09);江苏省高技术研究项目(BG2007324)

摘　　要：为了从单卵泡水平建立非闭锁大小卵泡颗粒细胞基因表达的消减文库,选择繁殖性能正常、空怀高繁殖力黄淮山羊2只,以氯前列烯醇处理40h后取卵巢,计数卵泡并测量卵泡直径,钝性分离采集卵泡颗粒细胞,置于液氮内保存,取样后的卵泡部分组织采用原位缺口末端标记法(TUNEL)进行闭锁鉴定。选择直径6和4mm非闭锁卵泡,提取颗粒细胞mRNA,扩增cDNA,监测抑制消减杂交及其文库构建的试验环节。结果表明:消减文库的阳性克隆测序成功率100%,从正向文库中随机挑取96个克隆进行差异表达的筛选,发现12个全新表达序列标签;应用RT-PCR对已知序列的抑制素βA亚基基因(INHBA)和谷胱甘肽S-转移酶A1基因(GSTA1)进行表达水平测定,证实了颗粒细胞2个基因mRNA的表达模式与差异显示的结果相同。表明消减文库构建成功。In order to construct a subtractive cDNA library of genes differentially expressed in follicular granulosa cells between a single large and small unatresia follicles of goat ovary,two Huanghuai goats with normally reproductive ability and high prolificacy during estrous cycle were selected,of which the ovaries were taken out after treating with prostaglandin 40 hours. Follicles were counted and measured,then granulosa cells of antral follicles were collected by blunt dissection and stored in liquid nitrogen. After sampling,part of the follicular tissue were collected to detect atresia by TdT-mediated dVTP nick end labeling （ TUNEL） method,and mRNA of granulosa cells in 6 mm and 4 mm diameter of unatresia follicles was extracted respectively,cDNA was then amplified,the experiments of suppression subtractive hybridization （ SSH） and its library construction were monitored. The results showed that success rate of sequencing for the positive clones in SSH library was 100%. Ninety-six clones randomly picked from the forward library were screened for differential expression by dot blot analysis and twelve novel expressed sequence tags （ ESTs） were found. Expression of inhibin βA gene （ INHBA） and glutathione S-transferase A1 gene （ GSTA1） in the follicles were determined by RT-PCR,confirming the same expression pattern.

关 键 词：山羊 卵巢 颗粒细胞 基因表达 消减文库 

分 类 号：S814.8[农业科学—畜牧学] S826[农业科学—畜牧兽医]