绵羊卵巢组织细胞酵母双杂交cDNA文库的构建及鉴定  

Construction and identification of yeast two-hybrid cDNA library of ovine ovarian tissue cells

作　　者：张丽萌 李闰婷[1,2] 聂晓宁 陈龙欣[2] 邢真真 刘爱菊[1] 房晓欢 韩红叶[1] 马润林 田树军 ZHANG Limeng;LI Runting;NIE Xiaoning;CHEN Longxin;XING Zhenzhen;LIU Aiju;FANG Xiaohuan;HAN Hongye;MA Runlin;TIAN Shujun(College of Animal Science and Technology,Hebei Agricultural University,Baoding 071000,China;Laboratory of Molecular Biology,Zhengzhou Normal University,Zhengzhou 450000,China;Research Center of Cattle and Sheep Embryonic Technology of Hebei Province,Baoding 071000,Chin)

机构地区：[1]河北农业大学动物科技学院,河北保定071000 [2]郑州师范学院分子生物学实验室,河南郑州450000 [3]河北省牛羊胚胎工程技术研究中心,河北保定071000

出　　处：《畜牧与兽医》2020年第8期45-49,共5页Animal Husbandry & Veterinary Medicine

基　　金：河北省羊产业技术体系(HBCT2018140202)。

摘　　要：试验旨在构建绵羊卵巢组织细胞的cDNA文库,为进一步研究绵羊卵巢中蛋白互作机制提供工具。本研究以不同发情周期的绵羊卵巢组织为材料,利用TRIzol试剂提取绵羊卵巢组织细胞总RNA,应用SMART技术经过长链PCR(LD-PCR)合成双链cDNA(ds cDNA),通过CHROMA SPIN^TM+TE-400柱纯化得到ds cDNA,并与线性pGADT7-Rec共转化酵母Y187感受态细胞。用同源重组的方式,在酵母细胞内构建绵羊卵巢组织细胞酵母双杂交cDNA表达文库。结果构建了含有3×108个重组子的绵羊卵巢组织cDNA文库,插入片段长度多数为500~2000 bp,重组率达96%,重组子中平均插入的片段长度为1000 bp,文库滴度为2×107cfu/mL,符合建库标准。The purpose of this study was to construct a cDNA library of ovine ovarian tissue cells and to provide a tool for further study of the mechanism of protein interaction in ovine ovary.Trizol reagent was used to extract total RNA from the ovine ovarian tissue cells of sheep in different estrous cycles.The SMART technology was adopted to synthesize double strand cDNA(ds cDNA)by LD-PCR.The ds cDNA was purified using the CHROMA SPIN^TM+TE-400 column,and were transformed into yeast Y187 cells together with linear pGADT7-Rec,and a yeast two-hybrid cDNA library of ovarian tissue cells was constructed by homologous recombination.The results showed that the constructed cDNA library of ovine ovary contained 3×108recombinants.The inserted cDNA fragments were 400 bp to 2500 bp long,and the recombination rate was 96%.The average length of the inserted fragments was 1000 bp,and the titers of cDNA library was as high as 2×107cfu/mL,which indicated that the present cDNA library met the criteria of library construction.

关 键 词：绵羊 卵巢 CDNA文库 酵母双杂交 

分 类 号：S814.8[农业科学—畜牧学]