番木瓜环斑病毒海南南瓜分离物全基因组序列分析
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摘要
【目的】研究海南南瓜上番木瓜环斑病毒(Papaya ringspot virus,PRSV)的全基因组特征及系统进化情况,为南瓜病毒病的综合防控提供依据。【方法】通过DAS-ELISA和RT-PCR等方法,检测采自海南澄迈桥头镇疑似感染PRSV的南瓜叶片中是否存在PRSV,采用分段扩增测序方法拼接获得PRSV-HnPumpkin基因组,利用NC-BI中的BLAST工具、DNAStar和MEGA 6.06软件进行序列分析及系统关系树构建。【结果】DAS-ELISA和RT-PCR检测结果表明,采自海南澄迈桥头镇疑似感染PRSV的南瓜叶片中存在PRSV(PRSV-HnPumpkin)。PRSV-HnPumpkin基因组全长为10 327nt,具有典型的PRSV基因组结构特征,含有1个开放阅读框,编码1个含有3 345个氨基酸的多聚蛋白。PRSV-HnPumpkin与已报道的其他PRSV分离物核苷酸和氨基酸之间的相似性分别为80.6%～95.1%和90.7%～97.9%。系统关系分析表明,PRSV-HnPumpkin与亚洲分离物处于同一组中,且与我国台湾分离物亲缘关系较近。【结论】获得了PRSV-HnPumpkin全序列,但其进化来源有待进一步探讨。
【Objective】The study clarified the genome characteristics of Papaya ringspot virus(PRSV)infecting pumpkin in Hainan and its phylogenetic relationship with reported PRSV isolates to provide basis for the comprehensive prevention of pumpkin virus disease.【Method】PRSV was detected from suspected virus-infected pumpkin leaf samples by DAS-ELISA and RT-PCR methods.The complete genomic sequence of PRSV-Hnpumpkin isolate was determined by assembling six fragments amplified from the sample by RT-PCR.Sequence was analyzed by BLAST and DNAStar.Neighbor-joining phylogram was constructed by MEGA 6.06.【Result】DAS-ELISA and RT-PCR confirmed the presence of PRSV(PRSVHnPumpkin)in the suspected virus-infected pumpkin leaf samples from Qiaotou,Chengmai county in Hainan.Its genome contained 10 327 nt with typical characteristics of previously reported PRSV isolates.The genome contained one ORFs,coding for one polyprotein with 3 345 amino acids.The identities of nucleotide and amino acids with other isolates in GenBank were 80.6%-95.1%and 90.7%-97.9%,respectively.Phylogram analysis revealed that PRSV-HnPumpkin and Asian isolates were in the same group,and it shared closer relationship with the isolates from Taiwan,China.【Conclusion】The complete sequence of PRSV-HnPumpkin was obtained,and further investigation is needed to identify its evolution source.
【Objective】The study clarified the genome characteristics of Papaya ringspot virus(PRSV)infecting pumpkin in Hainan and its phylogenetic relationship with reported PRSV isolates to provide basis for the comprehensive prevention of pumpkin virus disease.【Method】PRSV was detected from suspected virus-infected pumpkin leaf samples by DAS-ELISA and RT-PCR methods.The complete genomic sequence of PRSV-Hnpumpkin isolate was determined by assembling six fragments amplified from the sample by RT-PCR.Sequence was analyzed by BLAST and DNAStar.Neighbor-joining phylogram was constructed by MEGA 6.06.【Result】DAS-ELISA and RT-PCR confirmed the presence of PRSV(PRSVHnPumpkin)in the suspected virus-infected pumpkin leaf samples from Qiaotou,Chengmai county in Hainan.Its genome contained 10 327 nt with typical characteristics of previously reported PRSV isolates.The genome contained one ORFs,coding for one polyprotein with 3 345 amino acids.The identities of nucleotide and amino acids with other isolates in GenBank were 80.6%-95.1%and 90.7%-97.9%,respectively.Phylogram analysis revealed that PRSV-HnPumpkin and Asian isolates were in the same group,and it shared closer relationship with the isolates from Taiwan,China.【Conclusion】The complete sequence of PRSV-HnPumpkin was obtained,and further investigation is needed to identify its evolution source.
引文
[1]肖火根,范怀忠.植物组织粗汁液中的番木瓜环斑病毒的ELISA检测技术[J].中国病毒学,1994(3):249-255.Xiao H G,Fan H Z.Studies on the detection of Papaya ringspot virus in infected tissue of plant by ELISA[J].Virologica Sinica,1994(3):249-255.
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[3]卢雅薇,沈文涛,唐清杰,等.番木瓜环斑病毒海南分离株全基因组序列分析[J].病毒学报,2008,24(2):152-154.Lu Y W,Shen W T,Tang Q J,et al.Sequence analysis of the complete genome of Papaya ringspot virus Hainan isolate[J].Chinese Journal of Virology,2008,24(2):152-154.
[4]阮小蕾,王加峰,李华平.VIGS介导的转复制酶基因番木瓜对PRSV的抗性[J].华中农业大学学报,2009,28(4):418-422.Ruan X L,Wang J F,Li H P.Virus-induced gene silencingmediated viral resistance of transgenic papaya to Papaya ringspot virus(PRSV)[J].Journal of Huazhong Agricultural University,2009,28(4):418-422.
[5]Zhang Y,Yu N,Huang Q,et al.Complete genome of Hainan Papaya ringspot virus using small RNA deep sequencing[J].Virus Genes,2014,48(3):502.
[6]韩杰,郑继平,言普,等.原核表达番木瓜环斑病毒NIaPro及其双重酶活鉴定[J].热带作物学报,2016,37(4):722-727.Han J,Zheng J P,Yan P,et al.Prokaryotic expression and biological activity of NIa-Pro protein fromPapaya ringspot virus[J].Chinese Journal of Tropical Crops,2016,37(4):722-727.
[7]Zhao H,Jia R Z,Zhang Y L,et al.Geographical and genetic divergence among Papaya ringspot virus populations within Hainan province,China[J].Phytopathology,2016,106(8):937.
[8]古勤生,Piero Roggero,Marina Ciuffo,等.我国北方地区葫芦科作物病毒病的调查与病原鉴定[J].云南农业大学学报,2003,18(4):88-89.Gu Q S,Piero R,Marina C,et al.The occurrence of cucurbit viruses in northern China[J].Journal of Yunnan Agricultural University,2003,18(4):88-89.
[9]Liao Y M,Gan X J,Chen B,et al.First report of Papaya ringspot virus and Zucchini yellow mosaic virus in Luohanguo(Siraitia grosvenorii)in China[J].Plant Disease,2005,89(5):530.
[10]尤毅,李华平,谢大森.侵染冬瓜的病毒病原种类鉴定[J].植物保护,2016,42(2):182-186.You Y,Li H P,Xie D S.Identification of pathogenic viruses infecting wax gourd[J].Plant Protection,2016,42(2):182-186.
[11]杨国慧,张仲凯,崔崇士.番木瓜环斑病毒南瓜分离物外壳蛋白基因的克隆及序列分析[J].植物保护学报,2007,34(3):273-276.Yang G H,Zhang Z K,Cui C S.Cloning and sequence analysis of CP genes of PRSV from pumpkin[J].Acta Phytophylacica Sinica,2007,34(3):273-276.
[12]朱静,谭冠林,包改丽,等.云南番木瓜环斑病毒的发生及遗传多样性[J].微生物学通报,2014,41(6):1142-1151.Zhu J,Tan G L,Bao G L,et al.Occurrence and genetic biodiversity of Papaya ringspot virus in Yunnan[J].Microbiology China,2014,41(6):1142-1151.
[13]Cheng D J,Huang X D,Zhang J W,et al.First report of Papaya ringspot virus associated with a ringspot disease of zucchini in northern China[J].Plant Disease,2017,101(5):847.
[14]黄显德,王玉,闫志勇,等.番木瓜环斑病毒山东分离物的全基因组序列分析[J].植物病理学报,2018,48(2):285-288.Huang X D,Wang Y,Yan Z Y,et al.Complete genomic sequence analysis of a Papaya ringspot virus isolate from Shandong province of China[J].Acta Phytopathologica Sinica,2018,48(2):285-288.
[15]Inoue-Nagata A K,Franco C D M,Martin D P,et al.Genome analysis of a severe and a mild isolate of Papaya ringspot virus-type W found in Brazil[J].Virus Genes,2007,35(1):119-127.
[16]Maina S,Coutts B A,Edwards O,et al.Papaya ringspot virus populations from East Timorese and northern Australian cucurbit crops:biological and molecular properties,and absence of genetic connectivity[J].Plant Disease,2017,101(6):985-993.
[17]Ali A.First complete genome sequence of Papaya ringspot virus-W isolated from a gourd in the United States[J].Genome Announcements,2017,5(2):e01434-16.
[18]Noa-Carrazana J C,González-De-León D,Silva-Rosales L.Molecular characterization of a severe isolate of Papaya ringspot virus in Mexico and its relationship with other isolates[J].Virus Genes,2007,35(1):109.
[19]Romay G,Lecoq H,Desbiez C.Zucchini tigre mosaic virus is a distinct potyvirus in the Papaya ringspot virus cluster:molecular and biological insights.[J].Archives of Virology,2014,159(2):277-289.
[20]Ortiz-Rojas L Y,Chaves-Bedoya G.Molecular characterization of two Papaya ringspot virus isolates that cause devastating symptoms in Norte de Santander,Colombia[J].European Journal of Plant Pathology,2017,148(4):1-12.
[21]陈涵.葫芦科作物种子中番木瓜环斑病毒的分子检测[D].福州:福建农林大学,2006.Chen H.Molecular detection of Papaya ringspot virus in seeds of cucurbitaceae plant[D].Fuzhou:Fujian Agriculture and Forestry University,2006.
[2]蔡建和,范怀忠.华南番木瓜病毒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.Cai J H,Fan H Z.Investigation of papaya virus disease and identification of Papaya ringspot virus strains in south China[J].Journal of South China Agricultural University,1994,15(4):13-17.
[3]卢雅薇,沈文涛,唐清杰,等.番木瓜环斑病毒海南分离株全基因组序列分析[J].病毒学报,2008,24(2):152-154.Lu Y W,Shen W T,Tang Q J,et al.Sequence analysis of the complete genome of Papaya ringspot virus Hainan isolate[J].Chinese Journal of Virology,2008,24(2):152-154.
[4]阮小蕾,王加峰,李华平.VIGS介导的转复制酶基因番木瓜对PRSV的抗性[J].华中农业大学学报,2009,28(4):418-422.Ruan X L,Wang J F,Li H P.Virus-induced gene silencingmediated viral resistance of transgenic papaya to Papaya ringspot virus(PRSV)[J].Journal of Huazhong Agricultural University,2009,28(4):418-422.
[5]Zhang Y,Yu N,Huang Q,et al.Complete genome of Hainan Papaya ringspot virus using small RNA deep sequencing[J].Virus Genes,2014,48(3):502.
[6]韩杰,郑继平,言普,等.原核表达番木瓜环斑病毒NIaPro及其双重酶活鉴定[J].热带作物学报,2016,37(4):722-727.Han J,Zheng J P,Yan P,et al.Prokaryotic expression and biological activity of NIa-Pro protein fromPapaya ringspot virus[J].Chinese Journal of Tropical Crops,2016,37(4):722-727.
[7]Zhao H,Jia R Z,Zhang Y L,et al.Geographical and genetic divergence among Papaya ringspot virus populations within Hainan province,China[J].Phytopathology,2016,106(8):937.
[8]古勤生,Piero Roggero,Marina Ciuffo,等.我国北方地区葫芦科作物病毒病的调查与病原鉴定[J].云南农业大学学报,2003,18(4):88-89.Gu Q S,Piero R,Marina C,et al.The occurrence of cucurbit viruses in northern China[J].Journal of Yunnan Agricultural University,2003,18(4):88-89.
[9]Liao Y M,Gan X J,Chen B,et al.First report of Papaya ringspot virus and Zucchini yellow mosaic virus in Luohanguo(Siraitia grosvenorii)in China[J].Plant Disease,2005,89(5):530.
[10]尤毅,李华平,谢大森.侵染冬瓜的病毒病原种类鉴定[J].植物保护,2016,42(2):182-186.You Y,Li H P,Xie D S.Identification of pathogenic viruses infecting wax gourd[J].Plant Protection,2016,42(2):182-186.
[11]杨国慧,张仲凯,崔崇士.番木瓜环斑病毒南瓜分离物外壳蛋白基因的克隆及序列分析[J].植物保护学报,2007,34(3):273-276.Yang G H,Zhang Z K,Cui C S.Cloning and sequence analysis of CP genes of PRSV from pumpkin[J].Acta Phytophylacica Sinica,2007,34(3):273-276.
[12]朱静,谭冠林,包改丽,等.云南番木瓜环斑病毒的发生及遗传多样性[J].微生物学通报,2014,41(6):1142-1151.Zhu J,Tan G L,Bao G L,et al.Occurrence and genetic biodiversity of Papaya ringspot virus in Yunnan[J].Microbiology China,2014,41(6):1142-1151.
[13]Cheng D J,Huang X D,Zhang J W,et al.First report of Papaya ringspot virus associated with a ringspot disease of zucchini in northern China[J].Plant Disease,2017,101(5):847.
[14]黄显德,王玉,闫志勇,等.番木瓜环斑病毒山东分离物的全基因组序列分析[J].植物病理学报,2018,48(2):285-288.Huang X D,Wang Y,Yan Z Y,et al.Complete genomic sequence analysis of a Papaya ringspot virus isolate from Shandong province of China[J].Acta Phytopathologica Sinica,2018,48(2):285-288.
[15]Inoue-Nagata A K,Franco C D M,Martin D P,et al.Genome analysis of a severe and a mild isolate of Papaya ringspot virus-type W found in Brazil[J].Virus Genes,2007,35(1):119-127.
[16]Maina S,Coutts B A,Edwards O,et al.Papaya ringspot virus populations from East Timorese and northern Australian cucurbit crops:biological and molecular properties,and absence of genetic connectivity[J].Plant Disease,2017,101(6):985-993.
[17]Ali A.First complete genome sequence of Papaya ringspot virus-W isolated from a gourd in the United States[J].Genome Announcements,2017,5(2):e01434-16.
[18]Noa-Carrazana J C,González-De-León D,Silva-Rosales L.Molecular characterization of a severe isolate of Papaya ringspot virus in Mexico and its relationship with other isolates[J].Virus Genes,2007,35(1):109.
[19]Romay G,Lecoq H,Desbiez C.Zucchini tigre mosaic virus is a distinct potyvirus in the Papaya ringspot virus cluster:molecular and biological insights.[J].Archives of Virology,2014,159(2):277-289.
[20]Ortiz-Rojas L Y,Chaves-Bedoya G.Molecular characterization of two Papaya ringspot virus isolates that cause devastating symptoms in Norte de Santander,Colombia[J].European Journal of Plant Pathology,2017,148(4):1-12.
[21]陈涵.葫芦科作物种子中番木瓜环斑病毒的分子检测[D].福州:福建农林大学,2006.Chen H.Molecular detection of Papaya ringspot virus in seeds of cucurbitaceae plant[D].Fuzhou:Fujian Agriculture and Forestry University,2006.
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