海南番木瓜环斑病毒全长cDNA克隆及其侵染性克隆构建
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摘要
为了从分子水平上解析番木瓜环斑病毒(Papaya ringspot virus,PRSV)的致病机理,寻找广谱、有效的抗病毒新策略,培育具有应用前景的抗病新品种,利用RT-PCR和快速cDNA末端扩增技术(RACE)对引起番木瓜环斑病毒病的PRSV海南海口分离物(PRSV-HN2)的基因组cDNA进行了全序列测定。PRSV-HN2全长cDNA包含10 326 nt(不包括3′端的polyA,Gen Bank登录号为KF791028),能编码3 346个氨基酸。对PRSV-HN2基因组核苷酸序列及其推导的氨基酸序列进行了结构和功能分析,结果表明,其与其它23个PRSV分离物的核酸序列相似度达81%~91%,氨基酸相似度为88%~94%。利用酵母同源重组系统成功构建其侵染性克隆载体并通过农杆菌转化与侵染,最终获得其侵染性克隆。这为在分子水平上研究PRSV遗传变异、侵染及致病机理奠定理论基础。
In order to understand the pathogenesis mechanism of papaya ringspot virus at the molecular level, and to find a more broad-spectrum and effective antiviral strategy, therefore to cultivate new varieties with resistant promising, the complete genomic sequence of papaya ringspot virus isolated from Hainan island in China was cloned and determined by using RT-PCR and rapid-amplification of cDNA ends( RACE) techniques( Gen Bank accession number: KF791028). The full-length cDNA contains 10 326 nt( not including the 3' end of the poly(A)), a total of 3 346 amino acids are encoded, which was Compared with other PRSV isolates, the nucleotide sequence similarity was 81% to 91%, also the amino acid similarity was 88% to 94%. The full-length c DNA was cloned into plant expression vector, then the infectious clone was obtained by Yeast homologous recombination system and further transformed into Agrobacterium tumefaciens. The research of genetic variation PRSV, infection and pathogenesis at the molecular level will be benefit from this study.
In order to understand the pathogenesis mechanism of papaya ringspot virus at the molecular level, and to find a more broad-spectrum and effective antiviral strategy, therefore to cultivate new varieties with resistant promising, the complete genomic sequence of papaya ringspot virus isolated from Hainan island in China was cloned and determined by using RT-PCR and rapid-amplification of cDNA ends( RACE) techniques( Gen Bank accession number: KF791028). The full-length cDNA contains 10 326 nt( not including the 3' end of the poly(A)), a total of 3 346 amino acids are encoded, which was Compared with other PRSV isolates, the nucleotide sequence similarity was 81% to 91%, also the amino acid similarity was 88% to 94%. The full-length c DNA was cloned into plant expression vector, then the infectious clone was obtained by Yeast homologous recombination system and further transformed into Agrobacterium tumefaciens. The research of genetic variation PRSV, infection and pathogenesis at the molecular level will be benefit from this study.
引文
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[13]Attasart P,Charoensilp C,Kerbundit S,et al.Nucleotide sequence of a Thai isolate of Papaya ringspot virus type W[J].Acta Viro1,2002,46(4):241-246.
[2]蔡英卿,赖钟雄.番木瓜生物技术研究进展[J].江西农业大学学报,2003,25(3):429-434.
[3]蔡建和,范怀忠.华南番木瓜病毒病及环斑病毒株系的调查鉴定[J].华南农业大学学报,1994,15(4):13-17
[4]周鹏,郑学勤.病毒RNA复制酶-Nib基因转化番木瓜的研究[J].热带作物学报,1995,16(增刊):40-43.
[5]周鹏,郑学勤,陈向民.番木瓜环斑病毒外壳蛋白与核酸酶嵌合基因转化番木瓜胚状体的研究[J].热带作物学报,1995,16(增刊):31-35.
[6]Fater Youssef,Armelle Marais,Chantal Faure,Pascal Gentit and Thie rry Candresse Strategies to facilitate the development of uncloned or cloned infectious full-length viral c DNAs:Apple chlorotic leaf spot virus as a case study[J].Virology Journal,2011(8):488.
[7]Tuo Decai,Shen Wentao,Yan Pu,et al.Complete genome sequence of an isolate of Papaya leaf distortion mosaic virus from commercialized PRSV-resistant transgenic papaya in China[J].European Journal of Plant Pathology,2013,57(4):452-455.
[8]Anandalakshmi R,Pruss G J,Ge X.,et al.A viral suppressor of gene silencing in plants[J].Nat1 Acad Sci USA,1998,95(22):13 079-13 084.
[9]Arbatova J,Lehto K,Pehu E,et al.Localization of the Pl protein of potato Y poty virus in association with cytoplasmic inclusion bodies and in the cytoplasm of infected cells[J].J Gen Virol,1998,79(10):2 319-2 323.
[10]Levis C,Astier-Manifacier S.The 5'untranslated region of PVY RNA,even Located in an internal position,enables initiation of translation[J].Virus Genes,1993,7(4):367-379.
[11]Lu Y W,Shen W T,Zhou P,et al.Complete genomic sequence of a Papaya ringspot virus isolate from Hainan Island,China[J].Archives of Virology,2008,153(5):991-993(SCI).
[12]Atreya C D,Atreya P L,Thornbury D W,et al.Site-directed mutations in the potyvirus HC-Pro gene affect helper component activity,virus accumulation,and symptom expression in infected tobacco plants[J].Virology,1992,191(1):106-111.
[13]Attasart P,Charoensilp C,Kerbundit S,et al.Nucleotide sequence of a Thai isolate of Papaya ringspot virus type W[J].Acta Viro1,2002,46(4):241-246.