[1]张蓓,刘同坤,黄菲艺,等.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[J].南京农业大学学报,2017,(4):592-600.[doi:10.7685/jnau.201612034]
 ZHANG Bei,LIU Tongkun,HUANG Feiyi,et al.Subcellular localization and interaction proteins screening of flowering gene BcFT in non-heading Chinese cabbage[J].Journal of Nanjing Agricultural University,2017,(4):592-600.[doi:10.7685/jnau.201612034]
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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()
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《南京农业大学学报》[ISSN:1000-2030/CN:32-1148/S]

卷:
期数:
2017年4期
页码:
592-600
栏目:
出版日期:
2017-07-07

文章信息/Info

Title:
Subcellular localization and interaction proteins screening of flowering gene BcFT in non-heading Chinese cabbage
作者:
张蓓 刘同坤 黄菲艺 邵帅旭 吴小婷 侯喜林
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, 江苏 南京 210095
Author(s):
ZHANG Bei LIU Tongkun HUANG Feiyi SHAO Shuaixu WU Xiaoting HOU Xilin
State Key Laboratory of Crop Genetics and Germplasm Enhancement/Key Laboratory of Biology and Germplasm Enhancement of Horticultural Crops in East China, Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, China
关键词:
不结球白菜BcFT亚细胞定位酵母双杂交
Keywords:
non-heading Chinese cabbageBcFTsubcellular localizationyeast two hybrid
分类号:
S634.3
DOI:
10.7685/jnau.201612034
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pEZS-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pGBKT7-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cDNA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cDNA为模板进行基因全长克隆,构建pGADT7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pGBKT7-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:BcPPD5和BcVHA-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,BcPPD5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PsbP结构域;BcVHA-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:
[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 pEZS-NL-BcFT and analysed the spatial expression of BcFT by subcellular localization technique. We also constructed pGBKT7-BcFT bait vector,transformed into yeast strain Y2H Gold to verify self-activation and autotoxicity. When screening the yeast two hybrid cDNA library of non-heading Chinese cabbage,we found several fragments that interacted with BcFT. The full-length cDNA was cloned from non-heading Chinese cabbage to construct the pGADT7 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 pGBKT7-BcFT was confirmed to have no autoactivation and toxicity. Finally,we obtained two proteins that interacted with BcFT,BcPPD5 and BcVHA-E1. Bioinformatics analysis predicted that these two proteins were hydrophilic proteins and had no signal peptide. BcPPD5 protein had a transmembrane region,in the secondary structure,the largest was the irregular crimps,and there was a PsbP domain between the 163 and 296 amino acid residues. For BcVHA-E1,there was no transmembrane region and specific domain. [Conclusions]BcFT protein expressed in all components of cell. BcFT could interact with BcPPD5 and BcVHA-E1,the further analysis showed that it may affect flowering through photoperiodic pathway.

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备注/Memo

备注/Memo:
收稿日期:2016-12-20。
基金项目: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)
作者简介:张蓓,硕士研究生。
通信作者:侯喜林,教授,博导,主要从事蔬菜遗传育种和分子生物学研究,E-mail:hxl@njau.edu.cn。
更新日期/Last Update: 2017-07-07