玉米根部酵母双杂交cDNA文库的构建及评价

Construction and Evaluation of Yeast Two Hybrid cDNA Library of Maize Root

从玉米各生长时期的根部中提取总RNA后,再利用磁珠法从中分离纯化出mRNA,然后以mRNA为模板,反转录合成cDNA第一链,再在DNA聚合酶作用下,通过长距离PCR,扩增双链cDNA(dscDNA)。利用SMART技术,通过同源重组的方法,在酵母菌株AH109中构建了玉米根部全长cDNA文库。经检测,文库的转化率为1.76×107转化子/3μgpGADT7-Rec,文库滴度为1.17×106pfu/mL,重组率为95%。

The total RNA was isolated from each periods of growth maize root and mRNA was separated and purified using the SA-PMPs method. We used mRNA as template and synthesized the frist strand eDNA, and then obtained double strand cDNA by using LD-PCR approach. By using SMART technique and homologous reorganization method, we constructed the yeast two hybrid cDNA library in yeast AH109, which the conversion rate of the library was about 1.76×10^7 recombinants/3 μg pGADT7-Rec, the capacity of the library would be upto 1.17×10^6 pfu/mL, and the recombinant rate would be 95%.