大叶落地生根酵母cDNA文库的构建及KdSAHH基因自激活检测

Construction of Yeast Hybrid cDNA Library of Kalanchoe daigremontiana and Self-activation Detection of KdSAHH

大叶落地生根(Kalanchoe daigremontiana)主要以叶缘凹陷处产生不定芽的方式实现高效再生,是研究无性繁殖的模式植物。为筛选大叶落地生根不定芽发育过程中重要调控蛋白的互作因子,本研究提取大叶落地生根不同组织来源的植物RNA,利用RNA反转录5′末端交换机制(Switching mechanism at 5′end of RNA transcript,SMART)建库技术和双链特异性核酸酶(Duplex-specific nuclease,DSN)均一化处理技术,构建大叶落地生根酵母cDNA杂交文库,同时构建诱饵载体pGBKT7-KdSAHH,并用营养缺陷法检测其自激活活性。对文库进行质量检测的结果显示:文库滴度为3.2×10^(6) CFU·mL^(-1),文库总容量为4.8×10^(7) CFU,插入片段长度在250~2000 bp范围内,重组率为91.7%。诱饵载体pGBKT7-KdSAHH构建成功且在酵母中无转录自激活活性。本研究为后续利用酵母杂交技术筛选大叶落地生根KdSAHH基因的互作蛋白提供基础。

Kalanchoe daigremontiana achieves highly efficient regeneration by producing plantlets in the depression area of the leaf margin,which is a model plant for studying asexual reproduction.To screen out the interaction factors of important regulatory protein during adventitious bud development of K.daigremontiana,plant RNA was extracted from different tissues of K.daigremontiana in this study.A yeast hybrid cDNA library with a high capacity for K.daigremontiana was constructed by DSN-normalization method and SMART technique.At the same time,the bait vector pGBKT7-KdSAHH was constructed,and self-activation assays were performed by auxotrophic medium culture after sequencing detection.The cDNA yeast hybrid library of Kalanchoe daigremontiana was successfully constructed.The library quality test results showed that the library titration number was 3.2×10^(6) CFU·mL^(-1),and the library capacity was 4.8×10^(7) CFU,and the inserted fragment length ranged from 250 to 2000 bp,and the cDNA fragment recombination rate was 91.7%.The bait vector,pGBKT7-KdSAHH,was successfully constructed,which had no self-activation effect in yeast.This study could be used for further research of screening out interaction proteins of KdSAHH by yeast hybrid technology.