日本结缕草(Zoysia japonica)酵母杂交cDNA文库的构建及分析  

Construction and Analysis of A Yeast Hybrid cDNA Library of Zoysia japonica

作　　者：李艳茹 周丽莹 董笛 刘亚玲 李舒文 王梦迪 晁跃辉[1] LI Yan-ru;ZHOU Li-ying;DONG Di;LIU Ya-ling;LI Shu-wen;WANG Meng-di;CHAO Yue-hui(School of Grassland Science,Beijing Forestry University,Beijing 100083,China;School of Life Science,Beijing Institute of Technology,Beijing 100081,China;Inner Mongolia Pratacultural Technology Innovation Center Co.,Ltd,Hohhot,Inner Mongolia 010010,China)

机构地区：[1]北京林业大学草业与草原学院,北京100083 [2]北京理工大学生命学院,北京100081 [3]内蒙古草业技术创新中心有限公司,内蒙古呼和浩特010010

出　　处：《草地学报》2023年第11期3271-3277,共7页Acta Agrestia Sinica

基　　金：内蒙古草业技术创新中心有限公司项目(CCPTZX2023B04)资助。

摘　　要：酵母双杂交和单杂交技术是生物大分子互作和调控研究中的高效分子生物学技术,是蛋白质互作或蛋白质与DNA互作筛选的手段。本研究旨在构建高容量的日本结缕草(Zoysia japonica)酵母文库,以便后续深入挖掘日本结缕草相关基因,进行基因功能及基因网络研究。本研究应用不同组织来源以及不同生长时期的日本结缕草,通过SMART技术构建了高容量的酵母杂交cDNA文库。质量检测结果显示:次级文库库容为4.0×10^(6)cfu·mL^(-1),总克隆数为1.6×10^(7),重组率>95%。最终获得的Y187酵母文库工作液细胞密度为3×10^(7)cells·mL^(-1),插入片段具有良好的多态性。以日本结缕草类胡萝卜素裂解双加氧酶ZjCCD7作为诱饵,从构建的酵母文库中筛选到一个与ZjCCD7互作的蛋白ZjatpG,证明该文库质量较高,符合酵母杂交筛选试验标准,可适用于调控和互作蛋白筛选。本研究为筛选日本结缕草的互作蛋白以及基因功能研究提供了基础。Yeast two-hybrid and one-hybrid techniques are essential and efficient molecular biology instruments for examining biomolecular interactions and regulation,as well as for screening protein-protein or protein-DNA interactions.The objective of this research was to construct a high-capacity gene library for Zoysia japonica to facilitate further investigation of its related genes,the functions of its genes,and the gene networks.We constructed a high-capacity yeast hybrid cDNA library using the SMART technology and the distinct tissues and growth stages of Zoysia japonica.The quality evaluation on that library revealed a secondary library capacity reached 4.0×10^(6)cfu·mL^(-1),the total clones count for 1.6×10^(7),and the recombination rate was more than 95 percent in that library.The obtained Y187 yeast library had a cell concentration of 3×10^(7)cells·mL^(-1)in its working liquid.A gel electrophoresis analysis revealed an excellent polymorphism took place in the inserted fragments.The Zoysia japonica carotenoid cleavage dioxygenase 7(ZjCCD7)was used as a bait to test the constructed library,and the result showed that an interaction protein ZjatpG was screened from the yeast hybrid library.These results demonstrated that the constructed Zoysia japonica library had a high quality,and complied with the criteria for the yeast hybrid screening experiments.It is suitable for screening interacting proteins and researching gene function,laying the groundwork for identifying interacting proteins and researching on the function of target genes in Zoysia japonica.

关 键 词：日本结缕草 酵母文库 SMART技术 

分 类 号：S543[农业科学—作物学]