芥菜开花调控蛋白SVP与FLC酵母表达载体的构建及其相互作用研究  

Determination of Interactions Between SVP and FLC in Brassica juncea Coss. by Yeast Two-Hybrid System

作　　者：汤青林[1] 李念祖[1] 丁宁[1] 陈竹睿[1] 宋明[1] 王志敏[1] 

机构地区：[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　　处：《园艺学报》2012年第6期1175-1182,共8页Acta Horticulturae Sinica

基　　金：国家重点基础研究发展计划‘973’项目(2012CB113900);国家自然科学基金项目(31000908);重庆市自然科学基金项目(2011BA1002);中央高校基本科研业务费专项(XDJK2012B020)

摘　　要：为深入研究芥菜开花信号整合子的两个核心调节因子SHORT VEGETATIVE PHASE(SVP)与FLOWERING LOCUS C(FLC)相互作用的分子机理,通过PCR扩增,从芥菜材料‘QJ’中分别克隆含EcoRⅠ/BamHⅠ双酶切位点的SVP和FLC编码区全长,并利用酵母双杂交体系,将FLC与GAL4报告基因DNA激活域融合(pGADT7FLC),SVP与GAL4报告基因DNA结合域融合(pGBKT7SVP)。两种重组质粒分别转化酵母Y187和Y2HGold后未出现自激活和毒性现象。融合的二倍体酵母(pGADT7FLC×pGBKT7SVP)能在选择性固体培养基QDO/X/A(SD/-Ade/-His/-Leu/-Trp/X-α-Gal/AbA)上生长,并且菌落呈蓝色。将诱饵质粒(pGBKT7SVP)与猎物质粒(pGADT7FLC)载体互换(pGADT7SVP、pGBKT7FLC),再次转化酵母后仍能融合成二倍体酵母(pGADT7SVP×pGBKT7FLC),并同时激活报告基因AUR1-C、HIS3、ADE2、MEL1,由此表明SVP与FLC蛋白能够相互结合。The fate of the flowering signal integrators is determined by SHORT VEGETATIVE PHASE （SVP） and FLOWERING LOCUS C （FLC） . For further study on the mechanism of the mutual recognition between SVP and FLC in Brassicajuncea Coss. （Mustard） variety＇ QJ＇, the coding sequences of SVP and FLC with digestion sites of EcoR I/BamH I were respectively amplified via PCR, and the interactions between SVP and FLC were detected by the yeast two-hybrid system. The full-length FLC was fused to the GAL4 DNA activation domain, which was designated as pGADT7FLC and then transformed into Y187 yeast stain. While SVP was fused to the GAL4 DNA binding domain, which was designated as pGBKT7SVP and then transformed into Y2HGold yeast stain. The two transformed yeaststains did not exhibit autoactivation and toxicity. The yeast stains of pGADT7FLC and pGBKT7SVP could mate into yeast diploids. The zygote diploids grew on selective agar plates QDO/X/A （SD/-Ade/-His/-Leu/-Trp/X-a-Gal/AbA） with blue stains. The results strongly indicated that SVP and FLC could combine with each other. Furthermore, the expression vectors of bait plasmid （pGBKT7SVP） and prey plasmid （pGADT7FLC） were exchanged with each other. Then the recombinated yeast plasmids of pGADT7SVP and pGBKT7FLC were reconstructed and respectively transformed into Y187 and Y2HGold yeast stains. The yeast zygote diploids （pGADT7SVP×pGBKT7FLC） exhibit on selective agar plates QDO/X/A, and the DNA-BD and AD were brought into proximitry to activate transcription of four independent reporter genes （AUR1-C, HIS3, ADE2, MEL1 ）. The results showed that SVP and FLC could act with each other to combine and form a complex.

关 键 词：芥菜 SVP FLC 酵母双杂交 

分 类 号：S637.1[农业科学—蔬菜学]