《植物生理学报》 2015, 51(9): 1447-1456

白桦开花抑制因子BpFLC与BpSVP蛋白同源互作域筛选与分析

郑唐春, 臧丽娜, 代丽娟, 杨传平, 曲冠证^*

东北林业大学林木遗传育种国家重点实验室, 哈尔滨150040

通信作者：曲冠证；E-mail: quguanzheng@yahoo.com；Tel: 0451-82192695

摘　要：

开花是植物由营养生长向生殖生长的转换点, 该过程受内源基因与环境信号共同调控。FLC (Flowering locus C)和SVP (Short vegetative phase)是开花途径中关键的抑制开花因子。本文利用酵母双杂交技术深入研究白桦开花抑制因子FLC与SVP的自身聚合作用的分子机理。从重组质粒pGBKT7-BpFLC、pGBKT7-BpSVP分别克隆出6个BpFLC截短体[BpFLC1 (aa1~73)、BpFLC2 (aa1~173)、BpFLC3 (aa74~173)、BpFLC4 (aa60~208)、BpFLC5 (aa74~208)和BpFLC6 (aa174~208)]和6个BpSVP截短体[BpSVP1 (aa1~68)、BpSVP2 (aa1~172)、BpSVP3 (aa69~172)、BpSVP4 (aa60~225)、BpSVP5 (aa69~225)和BpSVP6 (aa173~225)], 分别编码MADS型蛋白的MI、MIK、K、IKC、KC和C域。在酵母Y2HGold菌中, 分别共转质粒pGBKT7-BpFLC1~6×pGADT7-BpFLC及pGBKT7-BpSVP×pGADT7-BpSVP1~6。酵母转化子Y2HGold [pGBKT7-BpFLC×pGADT7-BpFLC]、Y2HGold [pGBKT7-BpFLC2~5×pGADT7-BpFLC], 可在选择性固体培养基TDO (SD/-Leu/-Trp/-His)、QTO/A (SD/-Leu/-Trp/-His/-Ade/AbA)上生长, 并在QDO/A/X (SD/-Leu/-Trp/-His/-Ade/AbA/X-α-gal)上长出蓝色菌落, 表明BpFLC能与自身及截短体蛋白BpFLC2~5同源结合。此外酵母Y2HGold [pGBKT7-BpSVP×pGADT7-BpSVP]、Y2HGold [pGBKT7-BpSVP×pGADT7-BpSVP2~5]也能同时激活报告基因AUR1-C、HIS3、ADE2、MEL1, 说明BpSVP能与自身及截短体蛋白BpSVP2~5同源结合。对保守区的序列结构进一步分析发现: BpFLC与BpSVP的K域是它们能够同源结合, 介导BpFLC与BpSVP自身形成同源二聚体的关键。

关键词：白桦; BpFLC; BpSVP; 酵母双杂交; 截短体; 蛋白互作

收稿：2015-03-04   修定：2015-08-18

资助：国家高技术研究发展计划(2011AA100202-1-6)和“十二五”农村领域国家科技计划(2013AA102704)。

Identification and Analysis of Acting Domains Mediating the Protein Self Interactions Between BpFLC and BpSVP from Betula platyphylla

ZHENG Tang-Chun, ZANG Li-Na, DAI Li-Juan, YANG Chuan-Ping, QU Guan-Zheng^*

State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin 150040, China

Corresponding author: QU Guan-Zheng; E-mail: quguanzheng@yahoo.com; Tel: 0451-82192695

Abstract:

Flowering, transition from vegetative to reproductive phase in plants, is regulated by both endogenous and environmental signals. Flowering locus C (FLC) and short vegetative phase (SVP) are the key genes in delaying flowering. For further study on the mechanism of interaction between SVP and FLC in birch, the test was performed by yeast two-hybrid. The truncated genes of BpFLC [BpFLC1 (aa1~73), BpFLC2 (aa1~173), BpFLC3 (aa74~173), BpFLC4 (aa60~208), BpFLC5 (aa74~208) and BpFLC6 (aa174~208)] and BpSVP [BpSVP1 (aa1~68), BpSVP2 (aa1~172), BpSVP3 (aa69~172), BpSVP4 (aa60~225), BpSVP5 (aa69~225) and BpSVP6 (aa173~225)] were respectively cloned from yeast recombination plasmids pGBKT7-BpFLC and pGBKT7-BpSVP, the truncated forms respectively encoded the MI, MIK, K, IKC, KC and C domain of MADS protein. pGBKT7-BpFLC1~6×pGADT7-BpFLC and pGBKT7-BpSVP×pGADT7-BpSVP1~6 were co-transfered into yeast Y2HGold. The yeast strains of Y2HGold [pGBKT7-BpFLC×pGADT7-BpFLC] and Y2HGold [pGBKT7-BpFLC2~5×pGADT7-BpFLC] could grow on selective agar plates TDO (SD/-Leu/-Trp/-His), QTO/A (SD/-Leu/-Trp/-His/-Ade/AbA), or QDO/X/A (SD/-Leu/-Trp/-His/-Ade/AbA/X-α-gal) with blue stains. The results showed that BpFLC and truncated forms BpFLC2~5 protein or self-BpFLC could act with each other to form homodimers. Then Y2HGold [pGBKT7-BpSVP×pGADT7-BpSVP] and Y2HGold [pGBKT7-BpSVP×pGADT7-BpSVP2~5] were also brought into proximity to form protein compounds and activate transcription of four independent reporter genes, such as AUR1-C, HIS3, ADE2, MEL1. The results also showed that BpSVP and truncated forms BpSVP2~5 protein or self-BpSVP could act with each other to form homodimers. Further study of sequence structure of conserved domain, the results strongly showed that the K domain of BpFLC and K domain of BpSVP were the key structure domains and mediated the protein interactions between BpFLC and BpSVP protein homologous dimerization.

Key words: Betula platyphylla; BpFLC; BpSVP; yeast two-hybrid; truncated form; protein interaction