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

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 3.2×10⁶ CFU·mL^-1, and the library capacity was 4.8×10⁷ CFU, and the inserted fragment length ranged from 250 to 2 000 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.