《植物生理学报》 2013, 49(9): 935-942

甘蓝抽薹开花抑制因子FLC与SVP体外表达及相互作用

杨朴丽, 张丹华, 刘智宇, 许俊强, 王志敏, 汤青林*, 宋明*

西南大学园艺园林学院, 南方山地园艺学教育部重点实验室, 重庆市蔬菜学重点实验室, 重庆400715

通信作者：汤青林；E-mail: swutql@163.com, swausongm@163.com；Tel: 023-68250227, 023-68251093

摘　要：

甘蓝抽薹开花抑制因子FLC与SVP可能存在相互作用, 从而调节开花时间。为进一步的验证该相互作用, 以甘蓝‘ZQ’为材料, 扩增了594 bp的FLC cDNA和726 bp的SVP cDNA序列, 它们分别编码197和241个氨基酸, 且均属MIKC型蛋白。NCBI比对发现FLC与BoFLC3同源性高达98%, SVP与BoSVP-a同源性高达99%。将甘蓝FLC和SVP分别与原核表达载体pET43.1a融合, 构建重组质粒pET43.1a-FLC、pET43.1a-SVP, 并转化宿主菌大肠杆菌BL21, 均能够在体外表达目的蛋白。利用免疫共沉淀的原理及pET43.1a-FLC、pET43.1a-SVP融合蛋白序列中的6×His标签能与Ni+结合的特点, 结合SDS-PAGE, 检测到体外表达蛋白FLC与SVP能相互作用并形成复合体, 这为深入研究FLC与SVP互作机理及探讨其与抽薹开花因子的相互作用提供了理论依据和技术基础。

关键词：甘蓝; FLC; SVP; 体外表达; 蛋白质相互作用

收稿：2013-05-17   修定：2013-07-03

资助：国家重点基础研究发展计划(2012CB113900)、国家自然科学基金(31000908)、重庆市自然科学基金(2011BA1002)、中央高校基本科研业务费专项(XDJK2012B020)和国家大学生创新项目(201210635045)。

In vitro Expression and on the Interactions between FLC and SVP from Repressors of Bolting in Brassica oleracea L.

YANG Pu-Li, ZHANG Dan-Hua, LIU Zhi-Yu, XU Jun-Qiang, WANG Zhi-Min, TANG Qing-Lin∗, SONG Ming∗

Chongqing Key Laboratory of Olericulture, Key Laboratory of Horticulture Science for Southern Mountainous Regions, Ministry of Education, College of Horticulture and Landscape Architecture, Southwest University, Chongqing 400715, China

Corresponding author: TANG Qing-Lin; E-mail: swutql@163.com, swausongm@163.com; Tel: 023-68250227, 023-68251093

Abstract:

There exists a possible interaction regulating flowering time of bolting repression factors between FLC and SVP in Brassica oleracea L. To prove the interactive mechanism between FLC and SVP furtherly, the protein interaction in vitro was testified in B. oleracea (Cabbage) ‘ZQ’. In this study, we gained the cDNA sequences of FLC with 594 bp and SVP with 726 bp, and encode 197 and 241 amino acid residues, respectively, and both are MIKC-type protein. NCBI blast draws that the homology of FLC with BoFLC3 up to 98%, and SVP with BoSVP-a up to 99%. The FLC and SVP genes of B. oleracea were inserted into the expression vector pET43.1a, constructed the recombinant plasmids pET43.1a-FLC and pET43.1a-SVP, respectively, then transformated into E. coli (BL21), and both expressed target protein in vitro. With co-immunoprecipitation and characteristic of 6×His tag in fusion protein of pET43.1a-FLC and pET43.1a-SVP which could combine with Ni+, we detected that the two fusion proteins can form a complex with each other via SDS-PAGE. This research could provide theoretical and technical bases for analyzing the mechanism of protein interactions between FLC and SVP in vitro, and for probing the interactions among bolting repression factors furtherly.

Key words: Brassica oleracea L.; FLC; SVP; in vitro expression; protein interaction