基于Gateway技术的低成本植物双分子荧光互补分析系统  

Low-cost gateway-compatible bimolecular fluorescence complementation assay system

作　　者：周洁[1] 王栩鸣[1] 陈斌[1,2] 陈娟[1,2] 杨勇[1] 余初浪[1] 严成其[1] 陈剑平[1] 

机构地区：[1]浙江省农业科学院病毒学与生物技术研究所省部共建国家重点实验室培育基地,农业部植物保护与生物技术重点实验室,浙江省植物病毒重点实验室,浙江杭州310021 [2]浙江师范大学化学与生命科学学院,浙江金华321000

出　　处：《浙江农业学报》2013年第5期1024-1030,共7页Acta Agriculturae Zhejiangensis

基　　金：国家自然科学基金项目(30900263;31101208);浙江省自然科学基金项目(LY12C14011);浙江省创新团队项目(2009R50032)

摘　　要：创制基于Gateway重组技术的双分子荧光互补(bimolecular fluorescence complementation,BiFC)载体,同时创制低成本低克隆背景的Gateway入门载体。利用入门载体,采用Gateway技术快速将目标基因重组到BiFC目标载体,并采用农杆菌注射侵染烟草的方法观察荧光互作结果。构建的Gateway入门载体,由于带有ccdB基因,非线性化的载体无法在ccdB敏感菌株中生长,因此消除了载体本身的背景。同时由XcmⅠ酶切后产生的T突出,可以很好地与PCR产物结合,连接效率和普通商业化T载体一致。此外,由于载体可由实验室自行制备且采用了TA克隆,创制入门克隆的成本大大降低。借助该研究改造的BiFC目标载体,目的基因能快速地重组到目的载体用于基因互作鉴定。利用本研究构建的Gateway系统,能够以较低成本快速的进行基因克隆和互作研究。A gateway-compatible bimolecular fluorescence complementation（BiFC） assay system as well as a low-cost vector for entry cloning was constructed. With newly designed entry vector and gateway technology, target genes were quickly recombined to the BiFC destination vectors and introduced to tobacco leaves with Agrobacterium-mediated injec- tion, and the interactive fluorescence under confocol microscope was observed. Gateway entry vector developed in this study has a very low background for gene cloning. Due to the ccdB gene, the nonlinear vector cannot survive in ccdB sensitive strains after transformation, thus eliminating the background of the vector. After the digestion of XcmI, the linearized entry vector has a T overhang at 3＇ , and the PCR product can be efficiently ligated to the vector as same as a commercial T vector. Therefore the vector developed in this study can be easily prepared in laboratory and applied to TA cloning, which greatly reduced the cost for entry cloning. With the BiFC destination vectors produced in this study, the target- gene can be quickly recombined to the destination vectors and used for gene interaction assay. With the gate- way system developed in this study, genes can be efficiently cloned and applied to interaction assay with a lower cost.

关 键 词：入门载体 BIFC 基因互作 GATEWAY 

分 类 号：Q78[生物学—分子生物学]