番茄斑驳花叶病毒海南分离物全基因组序列分析
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摘要
本研究采用小RNA深度测序及RT-PCR检测技术鉴定海南省陵水县冬种番茄(圣女果)病株受到烟草花叶病毒属的番茄斑驳花叶病毒(Tomato mottle mosaic virus,ToMMV)侵染。通过机械摩擦接种,将该病原病毒保存于本生烟植株上,命名为番茄斑驳花叶病毒海南分离物(ToMMV-HN)。采用RT-PCR分段扩增获得了该病毒分离物的全基因组序列。测序结果表明,该病毒分离物基因组全长6399 nt,含有4个开放阅读框,编码4种蛋白,与已报道的ToMMV9个分离物核苷酸和编码产物氨基酸序列相似性分别为98.0%～100%和99.2%～100%;与同属的番茄花叶病毒(Tomato mosaic virus,ToMV)、番茄棕色皱果病毒(Tomato brown rugose fruit virus,TBRFV)和烟草花叶病毒(Tobacco mosaic virus,TMV)代表分离物核苷酸序列相似性分别为84.7%、80.7%和79.0%。系统进化分析结果表明,ToMMV-HN与该病毒中国西藏和云南辣椒分离物亲缘关系最近,与美国加州和南卡州番茄分离物次之,与西班牙和巴西番茄分离物亲缘关系相对较远。本研究为我国番茄病毒病诊断和防控,尤其是抗病品种选育提供了基础资料。
Small RNA deep sequencing and RT-PCR techniques were used to detect the viral pathogen infecting cherry tomato(Lycopersicon esculentum) samples collected from Lingshui County, Hainan Province in 2016. Tomato mottle mosaic virus(ToMMV) was identified from the plants showing dwarf, leaf chlorotic and mottle symptoms. The virus was preserved on the Nicotiana benthamiana by mechanical inoculation, and named as tomato mottle mosaic virus Hainan isolate(ToMMV-HN). Then its whole genome sequence was obtained by RT-PCR. It was determined that the complete genome of ToMMV-HN was 6399 nt in length and contained 4 open reading frames. The identity of nucleotide and encoded amino acid of this isolate with other reported 9 ToMMV isolates was 98.0%–100% and 99.2%–100%, respectively. The identity of nucleotide with Tomato mosaic virus(ToMV), Tomato brown rugose fruit virus(TBRFV) and Tobacco mosaic virus(TMV) which belonging to the genus Tobamovirus was 84.7%, 80.7% and 79.0%, respectively. Phylogenetic analysis showed that ToMMV-HN shared the closest relationship with pepper isolates from Tibetan and Yunnan, China, followed by tomato isolates from California and South Carolina, USA, and relatively distant from Spanish and Brazilian tomato isolates of this virus. This is the first report of the whole genome sequence of ToMMV isolated from tomato in China, which would provide basic information for the diagnosis and prevention of tomato viral disease in China, especially for disease-resistant variety breeding.
Small RNA deep sequencing and RT-PCR techniques were used to detect the viral pathogen infecting cherry tomato(Lycopersicon esculentum) samples collected from Lingshui County, Hainan Province in 2016. Tomato mottle mosaic virus(ToMMV) was identified from the plants showing dwarf, leaf chlorotic and mottle symptoms. The virus was preserved on the Nicotiana benthamiana by mechanical inoculation, and named as tomato mottle mosaic virus Hainan isolate(ToMMV-HN). Then its whole genome sequence was obtained by RT-PCR. It was determined that the complete genome of ToMMV-HN was 6399 nt in length and contained 4 open reading frames. The identity of nucleotide and encoded amino acid of this isolate with other reported 9 ToMMV isolates was 98.0%–100% and 99.2%–100%, respectively. The identity of nucleotide with Tomato mosaic virus(ToMV), Tomato brown rugose fruit virus(TBRFV) and Tobacco mosaic virus(TMV) which belonging to the genus Tobamovirus was 84.7%, 80.7% and 79.0%, respectively. Phylogenetic analysis showed that ToMMV-HN shared the closest relationship with pepper isolates from Tibetan and Yunnan, China, followed by tomato isolates from California and South Carolina, USA, and relatively distant from Spanish and Brazilian tomato isolates of this virus. This is the first report of the whole genome sequence of ToMMV isolated from tomato in China, which would provide basic information for the diagnosis and prevention of tomato viral disease in China, especially for disease-resistant variety breeding.
引文
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[21]陶永萍,尹跃艳,陈旭,等.小RNA深度测序鉴定昭通市烟草脉斑病病毒[J].植物保护,2018,44(2):69-74.
[2]周雪平,钱秀红.侵染番茄的番茄花叶病毒的研究[J].中国病毒学,1996,11(3):268-276.
[3]张桂芝.番茄病毒病的综合防治[J].农业科技通讯,2011(2):135-136.
[4]赵黎明,李刚,刘永光,等.番茄褪绿病毒与番茄黄化曲叶病毒复合侵染的分子鉴定[J].中国蔬菜,2014(12):15-20.
[5]Li R,Gao S,Fei Z,et al.Complete genome sequence of a new tobamovirus naturally infecting tomatoes in Mexico[J].Genome Announcements,2013,1(5):e00794-13.
[6]Padmanabhan C,Zheng Y,Li R,et al.Complete genome sequence of a tomato-infecting Tomato mottle mosaic virus in New York[J].Genome Announcements,2015,3(6):e01523-15.
[7]Fillmer K,Adkins S,Pongam P,et al.Complete genome sequence of a Tomato mottle mosaic virus isolate from the United States[J].Genome Announcements,2015,3(2):e00167-15.
[8]Moreira S R,Eiras M,Chaves A L R,et al.Characterization of the Tomato mosaic virus infecting tomato in Sao Paulo State,Brazil[J].Fitopatologia Brasileira,2003,28(6):602-607.
[9]Turina M,Geraats B P J,Ciuffo M.First report of Tomato mottle mosaic virus in tomato crops in Israel[J].New Disease Reports,2016,33:1.
[10]Li Y,Wang Y,Hu J,et al.The complete genome sequence,occurrence and host range of Tomato mottle mosaic virus Chinese isolate[J].Virology Journal,2017,14(1):15.
[11]Zerbino D R,Birney E.Velvet:Algorithms for de novo short read assembly using de Bruijn[J].Genome Research,2008,18(5):821-829.
[12]郑轩,成巨龙,赵震,等.五种烟草病毒TMV、CMV、TEV、PVY及TVBMV的多重RT-PCR同步检测[J].植物病理学报,2011,41(2):146-153.
[13]何自福,虞皓,余雪松,等.应用RT-PCR检测番茄上的CMV和To MV[J].华南农业大学学报,2003,24(3):36-39.
[14]朱红娟,胡国君,范旭东,等.我国部分地区沙地葡萄茎痘相关病毒分离物外壳蛋白序列变异分析[J].植物保护学报,2014,41(2):169-175.
[15]陈姗姗,张志想,刘国杰,等.苹果茎痘病毒苹果分离物的基因组序列测定及分析[J].植物保护学报,2014,41(2):248-249.
[16]Che H Y,Luo D Q,Cao X R.First report of tomato mottle mosaic virus in tomato crops in China[J/OL].Plant Disease,2018,http://www.apsnet.org/doi.10.1094/PDIS-03-18-0538-PDN.First Look.
[17]万学闪,朱庆胜.海南省露地樱桃番茄栽培关键技术[J].农业与技术,2015,35(17):109-110.
[18]朱庆胜.陵水县樱桃番茄嫁接栽培技术[J].中国农技推广,2017,33(7):32-33.
[19]云步宇.樱桃番茄反季节栽培技术[J].农技服务,2010,27(7):843-844.
[20]任海龙,徐麟,陈积豪.海南冬季鲜食番茄设施栽培技术初探[J].蔬菜,2015(1):61-62.
[21]陶永萍,尹跃艳,陈旭,等.小RNA深度测序鉴定昭通市烟草脉斑病病毒[J].植物保护,2018,44(2):69-74.