三种葡萄病毒的RT-PCR检测和系统进化分析RT-PCR detections and phylogenetic studies on three viruses from grapevine
王建辉;刘建军;陈克玲;李洪雯;何建;关斌;
摘要(Abstract):
【目的】为了研究不同葡萄病毒病快速检测方法和四川分离株系统进化,【方法】比较3种葡萄总RNA提取方法,还优化了葡萄病毒病的RT-PCR检测方法。分别克隆了3种病毒四川分离株的部分基因组区域,并开展了系统进化分析。【结果】结果表明,改进硅胶颗粒吸附法快速从葡萄枝条的韧皮组织中提取了总RNA,并以葡萄18SRNA为内参基因,RT-PCR分别成功扩增出3种病毒的目的片段。把16个地理起源的GLRaV-2分离株的外壳蛋白基因(Coat protein,CP)分成5个系统进化组。而7个地理起源的GVB分离株CP同源性为79.1%~98.7%。此外,5个GVA四川分离株分别位于系统进化树的4个聚类群。【结论】GLRaV-2、GVB和GVA不同地理起源的分离株在核酸水平上具有变异性,其中GVA四川分离株之间具有显著的遗传差异,可能包括4种株系。
关键词(KeyWords): 葡萄卷叶伴随2型病毒;葡萄B病毒;葡萄A病毒;病毒外壳蛋白基因;病毒沉默抑制子基因
基金项目(Foundation): “十二五”四川省财政基因工程:果树生物技术研究与创新(2011JYGC05-018)
作者(Authors): 王建辉;刘建军;陈克玲;李洪雯;何建;关斌;
DOI: 10.13925/j.cnki.gsxb.2013.02.002
参考文献(References):
- [1]LEE JUNGMIN,MARTIN R.Influence of grapevine leafroll associ-ated viruses(GLRaV-2 and-3)on the fruit composition of OregonVitis vinifera L.cv.Pinot noir:Phenolics[J].Food Chemistry,2009,112:889-896.
- [2]GARAU R,CUGUSI M,DORE M,PROTA U.Investigations on theyield of‘Monica’and‘Italia’vines affected by legno riccio(stempitting)[J].Phytopathol Mediterr,1985,24:64-67.
- [3]MENG B,LI C,GOSZYNSKI D E.Genome sequences of two bio-logically distinct strains of grapevine leafroll-associated virus 2 andsequence analysis[J].Virus Genes,2005,31:31-41.
- [4]SHI Bu-jun,HABILI N,GAFNY R.Extensive variation of sequencewithin Isolates of grapevine Virus B[J].Virus Genes,2004,29:279-285.
- [5]GOSZCZYNSKI D E,PREEZ J D,BURGER J T.Molecular diver-gence of Grapevine virus A(GVA)variants associated with Shirazdisease in South Africa[J].Virus Research,2008,138:105-110.
- [6]LIU Xiao,CHEN Jian,WANG Jian-hui,LIU Jian-jun.Identifica-tion of virus diseases in some gr apevine cultivar s and evaluation oftheir sanitary state[J].Journal of Fruit Science,2006,23(6):846-849.刘晓,陈建,王建辉,刘建军.部分葡萄品种的病毒病鉴定及健康状况评价[J].果树学报,2006,23(6):846-849.
- [7]PAN Zhi-yong,ZHAI Heng,ZUO Fang-mei,SHI Qi-zhao.The in-vestigation of the main viruses occurrences in grapevine productionareas at Shangdong[J].Plant Protection,2002,28(6):40-42.潘志勇,翟衡,左方梅,史启照,山东葡萄产区主要病毒病发生危害调查[J].植物保护,2002,28(6):40-42.
- [8]LIU Yong-qing,WANG Guo-ping.Techniques for detecting grapevinevirus[J].Sino-overseas Grapevine&Wine,2004(4):27-28.刘永清,王国平,葡萄病毒种类调查与检测技术研究[J].中外葡萄与葡萄酒,2004(4):27-28.
- [9]SAMBROOK J,FRITSCHE F,MANIATIS T.Molecular cloning:alaboratory manual[M].2nd.NY:Clod Spring Harbor Laboratory,1989.
- [10]ROYOJI N,MASAAKI N.Efficient methods for sample processingand cDNA synthesis byRT-PCR for the detection of grapevine virus-es and viroids[J].Journal of Virological Methods,2006,134:244-249.
- [11]JIANG Yu,WANG Jian-hui,YANG Hui,XU Mo-yun,YUANShu,SUN Wen,XU Wei-lin,XI De-hui,LIN Hong-hui.Identifi-cation and sequence analysis of turnip mosaic virus infection crucif-erous crops in southwest of China[J].Journal of Plant Pathology,2010,92:241-244.
- [12]FOISSAC X,SVANELLA-DULUCQ M J,CANDRESSE T,GEN-TIT P.Polyvalent detection of fruit tree tricho,capillo and foveav-iruses by nested RT-PCR using degenerated and inosine containingprimers(PDO RT-PCR)[J].Acta Hort,2000,550(5):37-44.
- [13]WANG Jian-hui,LIU Jian-jun,CHEN Ke-ling,LI Hong-wen,HEJian,GUAN Bin.Studies on the transgenic strawberry mediated byagrobacterium[J].Southwest China Journal of Agricultural Sciences,2012,25:1011-1013.王建辉,刘建军,陈克玲,李洪雯,何建,关斌.根瘤农杆菌介导草莓转基因研究[J].西南农业学报,2012,25:1011-1013.
- [14]WANG Jian-hui,LIU Xiao,XI De-hui,YUAN Shu,JIANG Yu,YANG Hui,DU Jun-bo,ZHANG Zhong-wei,CHEN Ke-ling,LINHong-hui.Cloning and prokaryotic expression of CP gene ofgrapevine virus A Sichuan isolate[J].Acta Horticulturae Sinica,2008,35(7):967-972.王建辉,刘晓,席德慧,袁澍,蒋彧,杨辉,杜俊波,张中伟,陈克玲,林宏辉.葡萄A病毒四川分离物的外壳蛋白基因克隆与原核表达[J].园艺学报,2008,35(7):967-972.
- [15]XU Z Y,HONG N,XING B,WANG G P.Partial molecular char-acterization of a Chinese isolate of grapevine leafroll-associatedvirus 2 and production of antisera to recombinant viral proteins[J].Journal of Plant Pathology,2006,88(1):89-94.
- [16]BERTAZZON N,BORGO M,VANIN S,ANGELINI E.Geneticvariability and pathological properties of Grapevine Leafroll-associ-ated Virus 2 isolates[J].Eur J Plant Pathol,2010,127:185-197.
- [17]WANG Jian-hui,LIU Jian-jun,CHEN Ke-ling,LI Hong-wen,XIDe-hui,LIN Hong-hui.Optimizing multiple detection and phyloge-netic studies on grapevine leafroll associated virus-3 and grapevinevirus A[J].Acta Horticulturae Sinica,2011,38(12):2401-2410.王建辉,刘建军,陈克玲,李洪雯,席德慧,林宏辉.葡萄卷叶伴随3型病毒、葡萄A病毒多重检测优化研究与其系统进化分析[J].园艺学报,2011,38(12):2401-2410.
- [18]REN Fang,FAN Xu-dong,DONG Ya-feng,ZHANG Zun-ping,WANG Jiao-min.Cloning and molecular variation analysis of coatprotein gene of grapevine virus A[J].Molecular Plant Breeding,2012,10(5):568-574.任芳,范旭东,董雅凤,张尊平,王教敏.葡萄A病毒外壳蛋白基因克隆及分子变异研究[J].分子植物育种,2012,10(5):568-574.
- [19]WANG Z Q,HONG N,LIU Y,XU W X,WANG G P.Genetic vari-ability and population structure of Grapevine virus A in China basedon the analysis of the coat protein gene[J].Canadian Journal of PlantPathology,2011,33:227-233.
- [20]MUROLO S,ROMANZZI G,ROWHANI A,MINAFRA A,LANOTTE P,BRANZANTI M B,SAVINO V.Genetic variability andpopulation structure of Grapeivne virus A coat protein gene from nat-urally infected Italian vines[J].European Journal Plant Pathology,2007,120:137-145.