您的位置:首页  > 论文页面

早实枳酵母双杂交cDNA文库的构建及评价

发表时间:2013-10-15  浏览量:1308  下载量:348
全部作者: 孙雷明,张金智,胡春根
作者单位: 华中农业大学园艺林学学院,园艺植物生物学教育部重点实验室
摘 要: 为探索早实枳的成花分子机理及各成花相关蛋白之间的互作关系,利用5’末端RNA转录转换机制(switching mechanism at 5 end of the RNA transcript, SMART)技术成功构建了早实枳特定组织的cDNA文库,用于后续的酵母双杂互作蛋白筛选实验。通过对总RNA的分离纯化得到mRNA,再将其反转录为第一链cDNA,经长链聚合酶链式反应(long distance-polymerase chain reaction, LD-PCR)扩增获得双链cDNA. 然后,通过质粒的同源重组,在酵母AH109菌株中构建了早实枳特定组织的双杂文库。经检测,构建的文库容量为1.61×106克隆/文库,文库滴度为1.84×108 pfu/mL. 文库质量及多态性较好,为筛选分离成花相关蛋白及研究其互作关系奠定了基础。
关 键 词: 果树学;早实枳;酵母双杂交;文库;构建
Title: Construction and evaluation of yeast two-hybrid cDNA library of precocious trifoliate orange
Author: SUN Leiming, ZHANG Jinzhi, HU Chungen
Organization: Key Laboratory of Horticultural Plant Biology, Ministry of Education, College of Horticulture and Forestry Science, Huazhong Agricultural University
Abstract: To explore the molecular mechanism and interaction among the proteins of flowering in precocious trifoliate orange, switching mechanism at 5 end of the RNA transcript (SMART) technique was used to construct the cDNA library. The total RNA was isolated form special tissues and mRNA was purified. We used mRNA as template and synthesized the first strand cDNA, then we obtained double strand cDNA by using long distance-polymerase chain reaction (LD-PCR) amplification. The yeast two-hybrid library of precocious trifoliate orange was constructed in yeast AH109 by homologous reorganization method. The results showed that the library contained 1.61�06 independent clone/library, and had a titer of 1.84�08 pfu/mL. Furthermore, the quality and polymorphism of the library was good, which privided a foudation for screening the interaction proteins related to flowering and their interaction research.
Key words: pomology; precocious trifoliate orange; yeast two-hybridization; library; construction
发表期数: 2013年10月第19期
引用格式: 孙雷明,张金智,胡春根. 早实枳酵母双杂交cDNA文库的构建及评价[J]. 中国科技论文在线精品论文,2013,6(19):1824-1829.
 
0 评论数 0
暂无评论
友情链接