水稻草状矮化病毒外壳蛋白CP自身互作研究  

Self-interaction of coat protein in rice grassy stunt virus

作　　者：胡昱颛 程淑媛 王全兴 杜磊 张金艺 高欣语 刘冰[1] 蒋军喜[1] 熊桂红 HU Yuzhuan;CHENG Shuyuan;WANG Quanxing;DU Lei;ZHANG Jinyi;GAO Xinyu;LIU Bing;JIANG Junxi;XIONG Guihong(College of Agronomy,Jiangxi Agricultural University,Nanchang 330045,China)

机构地区：[1]江西农业大学农学院,江西南昌330045

出　　处：《江西农业大学学报》2025年第1期32-40,共9页Acta Agriculturae Universitatis Jiangxiensis

基　　金：国家自然科学基金项目(31960533);江西省自然科学基金项目(20192BAB214004)。

摘　　要：【目的】明确水稻草状矮化病毒(Rice grassy stunt virus,RGSV)外壳蛋白CP的自身互作,为揭示CP蛋白在RGSV侵染中的功能提供理论依据。【方法】利用酵母双杂交(Y2H)技术鉴定RGSV CP与CP蛋白之间的互作。提取感染RGSV水稻叶片的总RNA,通过RT-PCR扩增得到CP基因。将CP基因分别构建到酵母表达载体pGBKT7和pGADT7上,利用菌液PCR鉴定阳性克隆。将酵母质粒组合pGBKT7-CP/pGADT7、pGBKT7-CP/pGADT7-CP、阳性对照pGADT7-T/pGBKT7-53、阴性对照pGADT7-T/pGBKT7-Lam分别转化到酵母感受态细胞Y2HGold中,先后涂布于缺陷型培养基SD/-Leu/-Trp和SD/-Ade/-His/-Leu/-Trp/X-α-gal上。通过观察酵母细胞在缺陷培养基上的生长和显色情况鉴定CP蛋白的毒性、自激活活性和自身互作关系。利用亚细胞定位和双分子荧光互补(BiFC)鉴定CP在本氏烟中的定位及互作。将CP构建到植物瞬时表达载体pEarleyGate101(101)、pEarleyGate202-YN(YN)、pEarleyGate202-YC(YC)上,并利用菌液PCR鉴定阳性克隆。将阳性克隆101-CP、YN-CP、YC-CP、空载体YN、YC分别转化到农杆菌GV3101中。将含阳性质粒101-CP的农杆菌单独注射本氏烟叶片;将含阳性质粒YN-CP/YC-CP、阴性对照YN-CP/YC、YN/YC-CP的农杆菌共注射本氏烟叶片;利用激光共聚焦显微镜观察融合蛋白的发光和定位。【结果】RT-PCR扩增得到CP基因,其大小为978bp。成功构建酵母表达载体pGBKT7-CP和pGADT7-CP。含质粒pGBKT7-CP/pGADT7、pGBKT7-CP/pGADT7-CP、阳性对照和阴性对照的酵母菌均能在缺陷培养基SD/-Leu/-Trp平板上生长,且含质粒pGBKT7-CP/pGADT7的酵母菌的生长情况与对照一致,说明CP蛋白对酵母菌无毒性。但仅含质粒pGBKT7-CP/pGADT7-CP的酵母菌和阳性对照可在缺陷培养基SD/-Ade/-His/-Leu/-Trp/X-α-gal平板上生长并显色,表明CP蛋白无自激活活性,且CP蛋白在酵母细胞中能够自身互作。成功构建CP基因的植物表达载体;亚细胞定位结果显示,CP蛋白主[Objective]This study aims to clarify the self-interaction of the coat protein(CP)of Rice grassy stunt virus(RGSV),which will provide a theoretical basis for revealing the function of the CP in RGSV infection.[Method]The interaction between RGSV CP and CP protein was identified by yeast two-hybrid(Y2H)technique.Total RNA was extracted from rice leaves infected by RGSV.Specific primers were designed according to the CDS sequence of CP gene and CP was amplified by RT-PCR.The CP gene was constructed into yeast expression vectors pGBKT7 and pGADT7 separately,which were verified by PCR with universal primer.The yeast plasmids pGBKT7-CP/pGADT7,pGBKT7-CP/pGADT7-CP,positive control pGADT7-T/pGBKT7-53 and negative control pGADT7-T/pGBKT7-Lam were transformed into Y2H Gold competent cells,respectively.The cytotoxicity,self-activation and self-interaction of CP were detected according to the growth and color development on the DO Supplement medium SD/-Leu/-Trp,SD/-Ade/-His/-Leu/-Trp with X-a-gal.The CP gene was constructed into plant transient expression vectors pEarleyGate101(101)contained yellow fluorescent protein(YFP),pEarleyGate202-YN(YN),pEarleyGate202-YC(YC)fused with C-and N-terminal half of YFP using gateway recombination technology,which were identified by PCR with specific primers.The positive clones 101-CP,YN-CP,YC-CP,YN,YC were transformed into Agrobacterium tumefaciens strain GV3101.The A.tumefaciens strain GV3101 containing the positive plasmid 101-CP was infiltrated into the leaves of Nicotiana benthamiana and the localization of CP was observed by confocal microscope.The A.tumefaciens containing the positive plasmids YN-CP/YC-CP and the negative controls YN-CP/YC,YN/YC-CP were mixed in equal volumes.Then they were co-infiltrated into the epidermal leaves of N.benthamiana.The bimolecular fluorescence complementation(BiFC)assay was used to confirm the interaction between CP and CP in plant cells.[Result]The CP gene was amplified by RT-PCR,and its size was 978 bp.The yeast expression vectors pGBKT7-CP and pGAD

关 键 词：水稻草状矮化病毒 外壳蛋白 载体构建 酵母双杂交 亚细胞定位 双分子荧光互补 蛋白互作 

分 类 号：S511[农业科学—作物学] S435.111.49