新疆肉用绵羊颗粒细胞抑制素βA基因RNAi载体的构建及其干扰效率验证  

Construction of RNA interference vector for inhibin-βA gene(INHβA)and its interference identification in ovarian granular cells of mutton sheep in Xinjiang

作　　者：林杉[1] 陈宝麒[2] 田占伟[2] 刘贤侠[2] 杨华[3] 赵宗胜[2] 

机构地区：[1]石河子大学生命科学学院,新疆石河子832003 [2]石河子大学动物科技学院,新疆石河子832003 [3]新疆农垦科学院,新疆石河子832003

出　　处：《中国兽医学报》2015年第8期1345-1352,共8页Chinese Journal of Veterinary Science

基　　金：新疆兵团绵羊繁育生物技术重点实验室开放课题基金资助项目(2011KLS06)

摘　　要：抑制素βA基因(inhibin-βA,INHβA)的表达与绵羊(Ovis aries)的产羔数有一定关系。为了观察INHβA对绵羊发情性状的影响以及在目的基因沉默后,引起的发情性状相关激素变化的情况,本研究利用RNAi技术构建载体干扰INHβA的表达。根据GenBank上绵羊INHβA(GenBank:NW_004080167.1)mRNA的序列设计干扰片段,通过酶切和测序验证,成功构建了4个INHβA基因RNAi质粒载体PLLU2G-shINHβA-1(I-1)、PLLU2G-shINHβA-2(I-2)、PLLU2G-shINHβA-3(I-3)、PLLU2G-shINHβA-4(I-4)和1个空载体(赛亚公司的PLLU2G)阴性对照;在成功分离卵巢颗粒细胞并建立培养体系后,利用Lip2000介导干扰载体转染细胞,通过荧光定量RT-PCR检测干扰前后INHβA的表达量。结果显示,4个干扰载体的干扰效率依次为34%,58%,39%,19%,其中I-2对INHβA表达的沉默效果最好。通过卵巢注射干扰载体I-2,检测干扰后绵羊血清INHβA、卵泡刺激素(FSH)、黄体生成素(LH)和雌二酮(E2)的含量。结果显示,注射干扰载体后INHβA和FSH分别呈现明显的下降和上升波峰,而血清中LH和E2水平没有发生明显变化。Inhibin-A（INHβA）correlates with the litter size of sheep.To investigate the influence of INHβAon sheep estrous and the hormone level related to the estrous after the silence of target gene（INHβA）,vectors interfering the INHβAexpression were constructed by RNAi technology in the study.According to the sheep INHβA sequence（GenBank：NW_004080167.1）on GenBank,we designed the interference fragments,and after enzyme digestion and sequencing,we constructed RNAi plasmid vectors PLLU2G-shINHβA-1（I-1）,PLLU2G-shINHβA-2（I-2）,PLLU2GshINHβA-3（I-3）,PLLU2G-INHβA-4（I-4）,and seted the empty vector PLLU2 Gas the negative control.After ovarian granular cells were successfully separated and established cultured system,transfecting cells were mediated by Lip2000,and the INHβAexpression before and after the interference were tested by the quantitative RT-RCR.The results showed that the four vectors suppressed INHβA mRNA levels with a silencing efficiency of 34%,58%,39% and 19% respectively,I-2had the best interference efficiency.Then,we tested the content of serum INHβA,FSH,LH and E2 by directing intro-ovarian injection of the I-2.The results showed that the INHβA level dropped and the follicle-stimulating hormone（FSH）rose in serum,while luteinizing hormone（LH）and estrodiol（E2）level did not change.This study sets a good theoretical basis of researching the breeding properties and estrus properties of sheep,and the shRNA vector is hopefully used for promoting the fecundity of sheep in practice.

关 键 词：抑制素βA基因(INHβA) 绵羊 RNA干扰(RNAI) 卵巢颗粒细胞 

分 类 号：S814.1[农业科学—畜牧学]