苦瓜果实酵母双杂交文库构建及McRPF互作蛋白筛选

为研究苦瓜果实成熟的分子调控机制,以苦瓜自交系E12201-e1为材料,取不同成熟期苦瓜果肉组织等量混合,采用All-Direct方法构建酵母双杂交cDNA文库。经质量鉴定,该文库库容为1.25×10⁷ CFU,平均插入片段大于1 200 bp,阳性率为100%,文库质量较高,符合建库标准。同时,以苦瓜果实成熟关键调控因子McRPF为诱饵,构建诱饵表达载体pGBKT7-McRPF。经鉴定该诱饵蛋白无自激活活性。利用共转化法,从文库中筛选到29个初始阳性菌落,经过DNA测序和BLAST比对分析,最终筛选得到12个可能与McRPF互作的蛋白,为进一步探究McRPF调控苦瓜果实成熟的分子机制奠定了理论基础。

Construction of Yeast Two-Hybrid Library and Screening of McRPF Interacting Proteins in the Fruit of Bitter Gourd

For revealing the regulatory molecular mechanism of bitter gourd fruit ripening, the flesh tissue of inbred line E12201-e1 at different maturity stages was used to construct yeast two-hybrid cDNA library by All-Direct method. The library capacity was 1.25×10⁷ CFU (Colony-Forming Units), recombination rate was 100%, and the average length of inserted fragment was longer than 1 200 bp. The library has high quality and it was conformed to the standard of library construction. The bait vector pGBKT7-McRPF was constructed and it has no self-activation activity. Twenty-nine initial positive clones were screened from the library using the co-transformation method. After sequencing and BLAST comparison analysis, 12 proteins that could interact with McRPF were finally identified. This work provides an important basis for studying the regulation mechanism of McRPF on the fruit ripening of bitter gourd.