芒果MiFTs基因家族克隆及其酵母双杂诱饵载体的构建

Molecular Clone and Construction of Yeast Two-Hybrid Bait Vector for MiFTs Gene Family in Mango

成花诱导是开花植物生长发育过程中最为重要的一环,而开花整合因子Flowering Locus T(FT)在这一过程中起非常重要的调控作用。目前,FT基因调控芒果成花的作用机制尚不清楚。本研究以‘四季蜜芒’为材料,克隆验证了从转录组测序数据中挖掘获得的3个FT同源基因,命名为MiFT1、MiFT2和MiFT3。生物信息学分析显示,3个MiFTs基因编码区长度分别为588 bp、540 bp和516 bp,分别编码氨基酸196个、180个和172个,分子量分别为48.97 kD、44.71 kD和42.27 kD,等电点分别为4.99、5.06和5.06,3个基因均含有一个保守的PEBP结构域。将MiFT1、MiFT2和MiFT3基因定向克隆到酵母表达载体pGBKT7上,通过菌落PCR和测序进行验证,并成功转化酵母菌株Y2H Gold感受态细胞。对诱饵质粒酵母菌进行自激活和毒性检测,发现pGBKT7-MiFT1、pGBKT7-MiFT2和pGBKT7-MiFT3在酵母菌种均不具备自激活性和毒性。此酵母诱饵蛋白表达载体被成功构建,为后续蛋白互作筛选提供参考。

Flowering induction is the most important link in the growth and development for flowering plants,and the flowering integration factor Flowering Locus T(FT)plays a very important regulatory role in this process.However,the mechanism of FT gene regulation on mango flowering is still unclear.In this study,three FT homologous genes were cloned and ver ified from the transcriptome data of Mangifera indica L.cv'SiJiMi',named MiFT1,MiFT2 and MiFT3,respectively.Bioinformatics analysis showed that the open reading frame(ORF)of these genes were 588 bp,540 bp and 516 bp,encoding amino acid 196,180 and 180,respectively.The molecular weight was 48.97 kD,44.71 kD and 42.27 kD,and isoelectric point was 4.99,5.06 and 5.06,respectively.All the three FT proteins contained a conservative PEBP domain.The MiFT1,MiFT2 and MiFT3 genes were cloned into the yeast expression vector pGBKT7,and identified by bacterial PCR and sequencing.Three recombinant bait plasmids pG-BKT7-MiFT were transformed into yeast strain Y2H Gold competent cells.Self-activation and toxicity detection of bait plasmid yeast showed that pGBKT7-MiFT1,pGBKT7-MiFT2 and pGBKT7-MiFT3 without self-activation and toxicity in yeast strains.These yeast bait protein expression vectors were successfully constructed,which provided reference for the subsequent protein interaction screening.