百脉根AD-cDNA文库的构建及与结瘤因子受体NFR1相互作用蛋白的鉴定  

Construction of Lotus japonicus AD-cDNA Library and Identification of Proteins Interact with NFR1

作　　者：朱浩[1] 朱辉[1] 杨绮[1] 陈春芬[1] 张忠明[1] 

机构地区：[1]华中农业大学生命科学技术学院/农业微生物学国家重点实验室,武汉430070

出　　处：《华中农业大学学报》2007年第6期793-798,共6页Journal of Huazhong Agricultural University

基　　金：国家自然科学基金项目(30570056)资助

摘　　要：以豆科植物百脉根为材料,构建了AD-cDNA表达文库。其库容量达到每3μg DNA 2×106酵母转化子,空载率为12.5%,平均片段长度为1.5 kb。以百脉根结瘤因子受体激酶基因1(nod factor receptor kinase,NFR1)蛋白激酶结构域的DNA片段(NFR1-PK)为诱饵,利用酵母双杂技术,筛选与NFR1相互作用的基因。结果在含X-gal的四缺选择性培养基上筛选到120个克隆。经质粒抽取、PCR鉴定、回转酵母验证得到80个阳性克隆。对26个确定阳性克隆的外源片段进行测序和同源性分析,通过NCBI数据库比对分析鉴定含有JAB1、AT-RICH结构域等4个与NFR1-PK相互作用的不同基因。通过半定量RT-PCR观察在百脉根中这些基因的表达情况,其结果可为进一步研究NFR1基因的功能和调控机制提供材料。An AD-cDNA library was constructed by using materials from legume Lotus japonicus. The library transformation efficiency is 2 × 10^6 transformants per 3 μg DNA, the empty transformants without cDNA insert is 12.5% of total transformants. The library was screened with the protein kinase domain of NFR1 （NFR1-PK） as bait by yeast two hybrid approach and 120 positive clones were selected on SD/-Leu-Trp-His-Ade plates containing X-gal. 80 clones were further confirmed by rescuing the plasmid from yeast, amplifying the DNA insert by PCR and retesting the phenotype. The positive clones were sequenced and searched for NCBI database by blast analysis. 4 genes interacted with NFR1-PK including JAB1 and ARID domain （AT-rich interacting domain）. To determine the expression of these genes in Lotus japonicus, the RT-PCR products of these genes were analyzed in different tissues. The result provided basis to study NFR1 function in Nod factor signal transduction.

关 键 词：百脉根 AD-cDNA文库 NFR1 酵母双杂交 

分 类 号：S154.381[农业科学—土壤学] Q785[农业科学—农业基础科学]