芥菜开花调控因子SVP与FLC蛋白互作的结构域筛选与鉴定  

Identification of Acting Domains Mediating the Protein Interactions Between SVP and FLC in Brassica juncea Coss.

作　　者：汤青林[1] 李念祖[1] 宋明[1] 丁宁[1] 陈竹睿[1] 刘智宇[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12期2395-2403,共9页Acta Horticulturae Sinica

基　　金：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：为阐明芥菜开花路径核心调节子SVP与FLC相互作用的结构域，从酵母重组质粒pGADT7SVP、pGBKT7FLC分别亚克隆了5个SVP截短体（SVPl～5）和5个FLC截短体（FLCl～5）。SVPI一5与FLCl～5编码蛋白的结构域均分别为MI、MIK、K、IKC和KC。利用酵母双杂交体系，分别构建酵母猎物质粒pGADT7SVPl—5与诱饵质粒pGBKT7FLCl～5，并转化对应的酵母Y187、Y2HGold菌。酵母转化子Y187[pGADT7SVP2～5]能与Y2HGold[-pGBKT7FLC]融合，并可在选择性固体培养基QDO／X／A上长出蓝色菌落，表明FLC能与截短体蛋白SVP2～5异源结合，SVP的K域（SVP3）可独立作用于FLC蛋白。此外，Y187[pGADT7SVP]×Y2HGold[pGBKT7FLC2～5]也能同时激活报告基因AURl．C、HIS3、ADE2、施z，，表明FLC的K域（FLC3）也可独立作用于SVP。进一步研究发现：Y187pGADT7SVP3]xY2Ht30ld[pGBKT7FLC3]正向杂交以及Y187I-pGADT7FLC3]×Y2HGoldl-pGBKT7SVP3]载体互换后杂交均可相互作用，表明SVP的K域（SVP第96～173位氨基酸区域）与FLC的K域（FLC第114～167位氨基酸区域）能够异源结合，是介导SVP与FLC蛋白互作的关键结构域。SVP and FLC were the key regulators in plant flowering pathways. For further study on the molecular mechanism of flowering control in Brassicajuncea Coss. （mustard）, the protein interaction domains between SVP and FLC were screened via yeast two-hybrid system. The truncated genes of SVP1- 5 and FLC1-5 were respectively subcloned from yeast recombination plasmids pGADT7SVP and pGBKT7FLC. The proteins encoded by SVP1-5 or FLC1-5 had MI-domain, MIK-domain, K-domain,IKC-domain and KC-domain, respectively. SVP1-5 truncated forms were fused into bait plasmid pGADT7, which were designated as pGADT7SVP1- 5 and then transformed into Y187 yeast stains. FLC1 -5 truncated forms were fused into prey plasmid pGBKT7, which were designated as pGBKT7FLC1- 5 and then transformed into Y2HGold stains. All the transformed yeast stains were not autoactivation and toxicity. The yeast stains of pGADT7SVP2- 5 and pGBKT7FLC could mate into zygotes and grew on selective agar plates QDO/X/A with blue stains. The results showed that SVP2- 5 truncated forms and FLC protein could act with each other to form heterodimers. K domain of SVP （ SVP3 ） was the key amino acid region to independently mediate the interactions between SVP and FLC. Then pGBKT7FLC2- 5 and pGADT7SVP were brought into proximity to form protein compounds and activate transcription of four independent reporter genes （A UR1-C, HIS3, ADE2, MEL1 ）. The resuRs also indicated that K domain of FLC （FLC3）played an important role in mediating the SVP-FLC heterodimers. To further.confirm the acting domains between SVP and FLC, the interactions between SVP3 and FLC3 were tested. The yeast zygotes （pGADT7SVP3 x pGBKT7FLC3, pGADT7FLC3 x pGBKT7SVP3） exhibit blue stains on selective agar plates QDO/X/A. The results strongly showed that the K domain of SVP （amino acid region from position 96 to 173） and the K domain of FLC （amino acid region from position 114 to 167） were the key structure domains and mediated the protein interactions between SVP and FLC.

关 键 词：芥菜 截短体 SVP FLC 酵母双杂交 

分 类 号：S637.1[农业科学—蔬菜学]