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

发布时间：2019-06-19 03:14

【摘要】：[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。
[Abstract]:[objective] flowering gene FT is the key gene downstream of flowering pathway. The purpose of this study was to study the subcellular localization of BcFT and the screening of interaction proteins in non-heading cabbage, and to understand the function and regulation mechanism of BcFT in non-heading cabbage. [methods] subcellular localization vector p EZS-NL-BcFT, was constructed to study the spatial expression of BcFT by subcellular localization technique. The protein interacting with BcFT was screened by yeast two-hybrid technique, and the yeast two-hybrid bait vector p GBKT7-BcFT, was constructed to transform yeast Y2H Gold strain, and the self-excited activity and autotoxicity were verified. The yeast two-hybrid c DNA library of non-heading cabbage was screened, and the fragments interacting with BcFT were obtained and compared according to the gene fragments. The full-length gene was cloned from non-heading cabbage c DNA as template, the p-GADT7 vector was constructed and verified by co-transformation. Finally, the two proteins were analyzed by bioinformatics. [results] BcFT protein was distributed in the whole cell. The bait vector p GBKT7-BcFT expression product has no self-excited activity and autotoxicity to yeast cells. Two proteins interacting with BcFT, Bc PPD5 and Bc VHA-E1., were obtained by yeast two-hybrid and co-transfer. Bioinformatics analysis predicted that the two proteins were hydrophilic and did not contain signal peptides. There was a transmembrane region in the amino acid sequence of Bc PPD5 protein, the proportion of irregular curl was the largest in the secondary structure, and there was a Psb P domain between 163 and 296 amino acid residues. Bc VHA-E1 protein had no transmembrane region, the proportion of 伪 helix in the secondary structure was the largest, and there was no specific domain. [conclusion] the expression of BcFT protein was confirmed in the whole cell. Two proteins interacting with BcFT were screened by yeast two-hybrid technique, and further analysis showed that they might affect flowering through photoperiod pathway.
【作者单位】：南京农业大学作物遗传与种质创新国家重点实验室/农业部华东地区园艺作物生物学与种质创新重点实验室;
【基金】：国家自然科学基金项目(31301782) 江苏省自然科学基金青年基金项目(BK20130673) 中国博士后科学基金项目(2014M550294);中国博士后科学基金项目(2015T80561)
【分类号】：S634.3

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