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园艺学报 ›› 2013, Vol. 40 ›› Issue (12): 2441-2452.

• 蔬菜 • 上一篇    下一篇

开花负调因子芥菜BjSVP与甘蓝BoFLC的异源互作研究

汤青林*, 刘智宇, 杨朴丽, 宋 明*, 王志敏   

  1. 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
  • 出版日期:2013-12-25 发布日期:2013-12-25

Identification of Protein Interactions Between BjSVP from Brassica juncea and BoFLC from Brassica oleracea

 TANG  Qing-Lin-*, LIU  Zhi-Yu, YANG  Pu-Li, SONG   Ming-*, WANG  Zhi-Min   

  1. (College of Horticulture and Landscape Architecture,Southwest University;Key Laboratory of Horticulture Science for
    Southern Mountainous Regions,Ministry of Education;Key Laboratory of Olericulture,Chongqing 400715,China)
  • Online:2013-12-25 Published:2013-12-25

摘要: 为阐明开花负调因子BjSVP(源于种子春化型作物芥菜)与BoFLC(源于绿体春化作物甘
蓝)异源聚合后在开花调控路径中的互作机制,从芥菜酵母重组质粒pGADT7-BjSVP 分别亚克隆含MI、
MIK、K、IKC、KC、IK、IK1L1K2L2、IK1L1K2、IK1L1、IK1、I 结构域的11 个SVP 截短体(BjSVPΔ1 ~ BjSVPΔ11),
构建猎物质粒pGADT7-BjSVPΔ1 ~ pGADT7-BjSVPΔ11 并转化酵母Y187 菌;从甘蓝中克隆了BoFLC 和
BoFLCzq 基因,构建诱饵质粒pGBKT7-BoFLC、pGBKT7-BoFLCzq 并转化酵母Y2HGold 菌。酵母双杂
交表明:芥菜BjSVP 能与甘蓝BoFLC 相互作用,在QDO/X/A 培养基上长出蓝色菌落,激活酵母报告基
因AUR1-C、HIS3、ADE2、MEL1。截短体BjSVPΔ2 ~ BjSVPΔ5 也能与BoFLC 相互作用,而BjSVPΔ7 ~
BjSVPΔ11 不能与BoFLC 互作。由此表明BjSVP 完整的K 域(BjSVP3)可独立作用于BoFLC,但K 域
亚域(K1、K2、K3)或者连接区(L1、L2)缺失突变后不能介导该作用。作用强度分析表明:BjSVP
的I 域能增强该蛋白互作,但M 域和C 域可能会干扰该作用;芥菜BjFLC 被甘蓝BoFLC 或BoFLCzq 替
换后,可明显增加作用强度;甘蓝FLC 的I 域第20 位、K 域第65 位和C 域第32 位氨基酸的变异很可能
与作用强度相关。

关键词: 芥菜, 甘蓝, FLC, SVP, 截短体, 酵母双杂交

Abstract: The transcription factors Flowering Locus C(FLC)and SHORT VEGETATIVE PHASE
(SVP)regulate the flowering time via protein interactions in the homologous plants of Brassica juncea or
Brassica oleracea,respectively. However,the heterologous protein-protein interactions between BoFLC of
Brassica oleracea and BjSVP of Brassica juncea have not been thoroughly understood. In an effort to
unravel the mechanisms involved in the heterologous interactions,we cloned BjSVPΔ1–BjSVPΔ11(MI,
MIK,K,IKC,KC,IK,IK1L1K2L2,IK1L1K2,IK1L1,IK1 or I domains)in Brassica juncea,BoFLC and BoFLCzq in Brassica oleracea,respectively. Then we tested the interactions between BoFLC and
BjSVP,using the Gal4 yeast two-hybrid system and the β-galactosidase activity assay. Results showed that
BjSVP or BjSVPΔ2 – BjSVPΔ5 interact with BoFLC. And the fused strains were incubated on
QDO/X-α-Gal/AbA plate and blue colonies were found,suggesting that the yeast fusion reporter genes
HIS3,AUR1-C,ADE2,and MEL1 were activated. It also indicated that the full length of K domain
(BjSVPΔ3)was the key amino acid region to independently mediate the protein interactions. However,
BjSVPΔ7–BjSVPΔ11 truncated forms without K1,K2,K3,L1 or L2 in K domain failed to act with
BoFLC. Furthermore,the heterologous interaction was enhanced by I domain of BjSVP,but weakened by
its M domain and C domain. The interaction also enhanced by BoFLC or BoFLCzq,compared with the
interaction of BjFLC protein. The three amino acid variations(site 20 of I domain,site 65 of K domain and
site 32 of C domain)were probably related to the protein interaction strength.

Key words: Brassica juncea, Brassica oleracea, FLC, SVP, truncated forms, yeast two-hybrid system

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