HbTCTP及其截短体酵母双杂交诱饵载体的构建及鉴定  

Construction and Identification of Yeast Two-hybrid Bait Vectors of HbTC-TP and Its Truncated Forms

作　　者：邓治[1] 胡燕玲 岳镒繁 代龙军[1] 杨洪[1] 李德军[1] Deng Zhi;Hu Yanling;Yue Yifan;Dai Longjun;Yang Hong;Li Dejun(State Key Laboratory Incubation Base for Cultivation and Physiology of Tropical Crops,Key Laboratory of Biology and Genetic Resources of Rubber Tree,Ministry of Agriculture and Rural Affairs,Rubber Research Institute,Chinese Academy of Tropical Agriculture Sciences,Haikou,571101;AgronomyCollege,Nanjing Agricultural University,Nanjing,210095)

机构地区：[1]中国热带农业科学院橡胶研究所,农业农村部橡胶树生物学与遗传资源利用重点实验室,省部共建国家重点实验室培育基地-海南省热带作物栽培生理学重点实验室,海口571101 [2]南京农业大学农学院,南京210095

出　　处：《分子植物育种》2020年第16期5389-5394,共6页Molecular Plant Breeding

基　　金：中国热带农业科学院基本科研业务费专项资金(1630022019018)资助。

摘　　要：为进一步鉴定橡胶树翻译控制肿瘤蛋白(HbTCTP)与橡胶延伸因子1 (HbREF1)互作的分子区域,构建Hb TCTP及其截短体的诱饵载体。本研究利用RT-PCR技术从橡胶树胶乳中扩增出包含HbTCTP不同结构域的片段,克隆到p GBKT7表达载体,通过酶切、菌落PCR和测序鉴定后,转化酵母Y2HGold感受态细胞,检测诱饵蛋白是否具有自激活作用和毒性。结果显示,成功构建6个包含HbTCTP不同结构域的诱饵载体,且诱饵载体对酵母宿主细胞无毒性和无自主激活报告基因的功能。本研究结果为进一步筛选HbTCTP与HbREF1互作的分子区域及阐明HbTCTP的功能提供科学依据。In order to further identifythe interactingdomains between translationallycontrolled tumor protein(Hb-TCTP)and rubber elongation factor(HbREF1)from Hevea brasiliensis,the bait vectors of HbTCTP and its trunca-ted forms were constructed.In this study,the fragments containing different functional domains of HbTCTP were amplified from latex of H.brasiliensis by RT-PCR and separately subcloned into the expression vector pGBKT7.After being identified by double enzyme digestion,colony PCR and DNA sequencing,the bait plasmids were transferred into Y2HGold competent yeast cell to detect their self-activation and toxicity.The results showed that six bait vectors containing different domains of HbTCTP were successfully constructed,and the bait proteins had no toxicity to yeast cells and no self-activation activity to the reporter genes.The bait vectors of HbTCTP and its truncated forms can be used for screening the interaction domain between HbTCTP and HbREF1.The result of this study might provide a scientific basis for further screening the interaction domain between HbTCTP and HbREF1 and elucidating the function of HbTCTP.

关 键 词：橡胶树(Hevea brasiliensis) HbTCTP 截短体 诱饵载体 酵母双杂交 

分 类 号：Q943.2[生物学—植物学] S794.1[农业科学—林木遗传育种]