大豆高盐胁迫下根部组织酵母双杂交文库的构建与分析  

Construction and analysis of a yeast two-hybrid cDNA library of soybean root tissue under high salt stress

作　　者：何晓兰[1] 许玲[1] 徐照龙[1] 卫陪陪 刘晓庆[1] 黄益洪[1] 张大勇[1] 

机构地区：[1]江苏省农业生物学重点实验室,江苏省农业科学院农业生物技术研究所,南京210014 [2]南京农业大学生命科学学院,南京210095

出　　处：《上海农业学报》2013年第6期20-23,共4页Acta Agriculturae Shanghai

基　　金：国家自然科学基金资助项目(31101166);江苏省农业科技自主创新资金项目[cx(12)3068];江苏省自然科学基金(BK2010474);国家十二五科技支撑计划(2011BAD35B06-4-3)资助

摘　　要：为了高效研究大豆耐非生物胁迫的分子调控网络,运用SMART(Switching Mechanism at 5'End of the RNA Transcript)与DSN(Duplex-Specific Nulease)相结合的技术构建了栽培大豆高盐胁迫下根部组织的酵母双杂交cDNA文库。提取高质量的总RNA,利用Oligo(dT)_(16)引物反转录合成cDNA后,经DSN处理,然后利用Advantage 2聚合酶进行长片段PCR扩增,电泳检测显示,产物均一,无高丰度条带,片段范围在0.75~5.00 kb;RT-PCR结果显示,GmActin基因在均一化后表达明显降低,表明均一化效果较好;最后通过SMART同源重组交换技术在酵母菌株Y18 7中构建了酵母双杂交所需的cDNA文库。文库质量检测结果表明:cDNA文库的总独立克隆数为5.61×10~6 cfu、文库滴度为3.4×10^(10)cfu/mL、重组率大于90%、插人片段长度为0.5~3.0 kb,主要集中在1.0 kb左右。In order to elucidate the molecular regulation networks for soybean tolerance to abiotic stresses,a yeast two-hybrid cDNA library from soybean root tissue under high salt stress was constructed using SMART （Switching Mechanism at 5＇ End of the RNA Transcript）combined with DSN（Duplex-Specific Nulease）technique.The high-quality total RNA was isolated and subjected to reverse transcription using Oligo（dT）16,and the double-strand cDNAs were synthesized and further treated with DSN,then long fragment PCR amplification was performed using Advantage 2 polymerase,electrophoresis results showed that the better homogeneous degree of PCR products ranged from 0.75-5.00 kb in length and the expression of GmActin significantly decreased by RT-PCR after homogenization.By using homologous recombination reaction,the dscDNAs were transformed into the Y187 yeast competent cell with the library plasmid pGADT7-Rec2 to construct the yeast two-hybrid cDNA library.Detection of the library quality demonstrated that it contained a total of 5.61 × 106 cfu independent yeast clones,the capacity of the library was up to 3.4 × 1010cfu/mL,the recombinant rate was more than 90%,and the average size of inserts was approximately 1.0 kb in the cDNA library.These results demonstrated that the library meet the requirements for screening the interaction proteins and this will lay the foundation for insight into the soybean functional genomics.

关 键 词：大豆 根 酵母双杂交文库 耐盐性 互作蛋白 

分 类 号：S565.1[农业科学—作物学] Q78[生物学—分子生物学]