不结球白菜开花基因BcFT的亚细胞定位及其互作蛋白的筛选

Subcellular localization and interaction proteins screening of flowering gene BcFT in non-heading Chinese cabbage

[目的]开花基因FT是开花通路下游关键基因。本文旨在研究不结球白菜BcFT的亚细胞定位及互作蛋白的筛选,深入了解不结球白菜BcFT的功能及其调控机制。[方法]构建亚细胞定位载体p EZS-NL-BcFT,利用亚细胞定位技术研究BcFT的空间表达。通过酵母双杂交技术筛选与BcFT互作的蛋白,构建酵母双杂交诱饵载体p GBKT7-BcFT,转化酵母Y2H Gold菌株,验证自激活性和自毒性。对不结球白菜酵母双杂交c DNA文库进行筛选,得到与BcFT互作的片段,并根据基因片段进行比对,以不结球白菜c DNA为模板进行基因全长克隆,构建p GADT7载体,进行共转化验证,最后对筛选得到的2个蛋白进行生物信息学分析。[结果]BcFT蛋白在整个细胞中均有分布。诱饵载体p GBKT7-BcFT表达产物对酵母细胞无自激活性和自毒性,通过酵母双杂交及共转验证得到2个与BcFT互作的蛋白:Bc PPD5和Bc VHA-E1。生物信息学分析预测这2个蛋白均为亲水性蛋白,不含信号肽,Bc PPD5蛋白的氨基酸序列存在1个跨膜区,二级结构中不规则卷曲所占比例最大,第163和296氨基酸残基之间存在1个Psb P结构域;Bc VHA-E1蛋白无跨膜区,二级结构中α螺旋所占比例最大,无特定结构域。[结论]确定了BcFT蛋白在整个细胞中均有表达。利用酵母双杂交技术筛选得到2个与BcFT互作蛋白,进一步分析发现其可能通过光周期路径影响开花。

[Objectives]FT is a key component in flowering pathways. To better understand the characteristics and molecular mechanisms of BcFT,we investigated the subcellular localization and interaction proteins of flowering gene BcFT. [Methods]We constructed the subcellular localization vector p EZS-NL-BcFT and analysed the spatial expression of BcFT by subcellular localization technique. We also constructed pGBKT7-BcFT bait vector,transformed into yeast strain Y2 H Gold to verify self-activation and autotoxicity. When screening the yeast two hybrid c DNA library of non-heading Chinese cabbage,we found several fragments that interacted with BcFT.The full-length c DNA was cloned from non-heading Chinese cabbage to construct the p GADT7 vectors. Then the bioinformatic analysis of the two proteins were carried out. [Results]BcFT protein was located in all components of cell. The expressed product of the bait vector p GBKT7-BcFT was confirmed to have no autoactivation and toxicity. Finally,we obtained two proteins that interacted with BcFT,Bc PPD5 and Bc VHA-E1. Bioinformatics analysis predicted that these two proteins were hydrophilic proteins and had no signal peptide. Bc PPD5 protein had a transmembrane region,in the secondary structure,the largest was the irregular crimps,and there was a Psb P domain between the 163 and 296 amino acid residues. For Bc VHA-E1,there was no transmembrane region and specific domain.[Conclusions]BcFT protein expressed in all components of cell. BcFT could interact with Bc PPD5 and Bc VHA-E1,the further analysis showed that it may affect flowering through photoperiodic pathway.