葡萄抗病毒双价RNAi植物表达载体构建及其对烟草的遗传转化Construction of binary RNAi expression vector with virus resistance and genetic transformation in tobacco
田莉莉,牛良
摘要(Abstract)：
【目的】为获得兼抗2种葡萄病毒的抗病植物新材料,【方法】利用Gateway技术成功构建了葡萄抗病毒双价RNAi表达载体。先采用RT-PCR分别克隆获得了葡萄卷叶病毒(GLRaV-3)和扇叶病毒(GFLV)外壳蛋白基因保守区段,用重叠延伸PCR方法串联获得了干扰片段GLRaV-GFLV(508 bp);通过TOPO克隆将该片段克隆入载体pENTR/SD/D构建了入门克隆载体pENTR/SD/D-GLRaV-GFLV;再经过LR反应使入门克隆载体pENTR/SD/D-GLRaV-GFLV上的干扰片段GLRaV-GFLV替换目标载体pH7GWIWG2(Ⅰ)上的ccdB基因,形成了含2种葡萄病毒基因片段的RNAi表达载体pH7GWIWG2(Ⅰ)-GLRaV-GFLV,用冻融法将其转入农杆菌菌株EHA105,以此作为工程菌进行转化烟草,【结果】经PCR鉴定证明目的片段已成功转入烟草中。【结论】研究基于Gateway技术的RNAi植物表达载体的构建和对烟草的遗传转化,为诱导转基因植物转录后基因沉默、获得抗2种病毒病的植物新材料提供了可能。
关键词(KeyWords)： 葡萄;抗病毒;RNAi植物表达载体;烟草;遗传转化
基金项目(Foundation): 国家自然科学基金资助项目(31071768)
作者(Author): 田莉莉,牛良
DOI: 10.13925/j.cnki.gsxb.2012.06.013
参考文献(References)：
- [1]CAI Wen-qi,XU Shao-hua,MANG Ke-qiang,MENG Bao-zhong,MA Yun-xia.Purification,serology and detection of grapevineleafroll virus[J].Acta Microbiologica Sinica,1997(5):385-392.蔡文启,徐绍华,莽克强,孟保中,马云霞.葡萄卷叶病毒的纯化、血清学研究及其在脱毒组培苗检测的应用[J].微生物学报,1997(5):385-392.
- [2]HE Pu-chao.Grape[M].Beijing:China Agriculture Press,1999:394-398.贺普超.葡萄学[M].北京:中国农业出版社,1999:394-398.
- [3]HILY RAVELONANDRO J M,DAMSTEEGT M,BASSETT V,PETRI C,LIU C,SCORZA R.Plum pox virus coat protein geneintron-hairpin-RNA(ihpRNA)constructs provide resistance toplum pox virus in Nicotiana benthamiana and Prunus domestica[J].Journal of the American Society for Horticultural Sciences,2007(132):850-858.
- [4]LI Qi-ke,TAO Gang,QIU You-bin,QIU Reng,CHI Sheng-qi,ZHU Ying,LIU Zuo-yi.RNAi vector targeting and interference ef-fect determination of PVY CP gene[J].Genomics and Applied Biolo-gy,2009,28(3):460-464.李奇科,陶刚,邱又彬,邱礽,迟胜起,朱英,刘作易.马铃薯Y病毒CP基因RNAi载体构建与干涉效果测定[J].基因组学与应用生物学,2009,28(3):460-464.
- [5]DING Yan-jie,ZHOU Zong-shan,DONG Ya-feng,XU Cheng-nan,WU Yu-xing,CHI Fu-mei.Construction of the RNAi vector ofGVA MP gene[J].Chinese Agricultural Science Bulletin,2011,27(10):256-259.丁艳杰,周宗山,董雅凤,徐成南,吴玉星,迟福梅.葡萄病毒AMP基因RNA干扰载体的构建[J].中国农学通报,2011,27(10):256-259.
- [6]SMITH N A,SINGH S P,WANG M B,STOUTJESDIJK P A,GREEN A G,WATERHOUSE P M.Total silencing by intron-spliced hairpin RNAs[J].Nature,2000,407:319-320.
- [7]KAMACHI S,MOCHIZUKI A,NISHIGUCHI M,TABEI Y.Trans-genic Nicotiana benthamiana plants resistant to cucumber greenmottle mosaic virus based on RNA silencing[J].Plant Cell Reports,2007,26(8):1283-1288.
- [8]GOUVEIA P,SANTOS M T,EIRAS-DIAS J E,NOLASCO G.Fivephylogenetic groups identified in the coat protein gene of grapevineleafroll-associated virus 3 obtained from Portuguese grapevine vari-eties[J].Archives of Virology,2011,156(3):413-420.
- [9]VIGNE E,DEMANGEAT G,KOMAR V,FUCHS M.Characteriza-tion of a naturally occurring recombinant isolate of Grapevine fanleafvirus[J].Archives of Virology,2005,150(11):2241-2255.
- [10]HORSH R B.A simple and general method for transferring genes intoplants[J].Science,1985,217:1229-1231.
- [11]YAN Pei-qiang,BAI Xian-quan,WAN Xiu-qing,GUO Zhao-kui,LI Li-jie,GONG Hai-ying,CHU Cheng-cai.Expression of TMVcoat protein gene RNAi in transgenic tobacco plants confer immunityto tobacco mosaic virus infection[J].Hereditas,2007,29(8):1018-1022.颜培强,白先权,万秀清,郭兆奎,李丽杰,公海英,储成才.应用RNAi技术培育抗TMV病毒转基因烟草[J].遗传,2007,29(8):1018-1022.
- [12]MA Jian,WEI Yi-fan,LI Zhi,WANG Pi-wu.Study on the con-struction method of plant RNAi expression vector[J].Journal of An-hui Agricultural Science,2009,37(18):8364-8366.马建,魏益凡,厉志,王丕武.植物RNA干扰表达载体构建方法的研究[J].安徽农业科学,2009,37(18):8364-8366.
- [13]LIU Ping,XIA Li-qiu,HU Sheng-biao,YAN Li,DING Xue-zhi,ZHANG You-ming,YU Zi-niu.Site-specific integration of heterol-ogous gene into Bacillus thuringiensis chromosome and its expression[J].Acta Microbiologica Sinica,2008,48(5):661-666.刘萍,夏立秋,胡胜标,严礼,丁学知,张友明,喻子牛.外源基因在苏云金杆菌染色体上的定点整合及表达[J].微生物学报,2008,48(5):661-666.