3个黑龙江烟区烟草花叶病毒分离物的全基因组序列测定与分析
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摘要
为明确黑龙江烟区烟草花叶病毒(Tobacco mosaic virus,TMV)分子株系和进化特征,从哈尔滨和牡丹江感病烟草采集到HEB1、HEB2和MDJ三个样品,通过RT-PCR扩增了其全基因组序列,并进行了一致率、重组、系统发育和选择压力等分析。结果表明,3个分离物全长均为6395核苷酸(GenBank登录号分别为MH595919、MH595920和MH595921)。HEB1和MDJ与辽宁分离物Beipiao(HE818412)一致率最高,分别为98.9%和99.6%;HEB2与云南分离物Chuxiong1(HE818417)一致率最高,为97.1%。HEB1是MDJ和HEB2的重组体。在根据全基因组序列构建的系统进化树中,TMV分为3个组,其中HEB1和MDJ均属Ⅰ(U1)组,HEB2属Ⅱ(Rakkyo)组。TMV的4个基因均处于负选择,其中衣壳蛋白基因的选择压力最大。
To elucidate the molecular strains and evolution characteristics of Tobacco mosaic virus(TMV) from tobacco plants in Heilongjiang producing area, three samples from Harbin and Mudanjiang tobacco fields were collected and designated as HEB1, HEB2 and MDJ. Their complete genomic fragments were amplified via RT-PCR. Sequence identities, recombination, phylogenetic relationship and selection pressure were analyzed. Results showed that the full-length genome of isolates HEB1, HEB2 and MDJ were 6395 nucleotides(nt)(GenBank accession numbers MH595919,MH595920 and MH595921). HEB1 and MDJ had the highest identities of 98.9% and 99.6%, respectively, with isolate Beipiao(HE818412) from Liaoning province, while MDJ had the highest identity of 97.1% with isolate Chuxiong1(HE818417) from Chuxiong of Yunnan province. HEB1 was a recombinant of isoaltes MDJ and HEB2. In the phylogenetic tree constructed with the complete genomic sequence, TMV was divided into three groups, among which HEB1 and MDJ belong to group Ⅰ(U1) while HEB2 belong to group Ⅱ(Rakkyo). All four proteins of TMV were under negative selection, with CP gene under the highest selection pressure.
To elucidate the molecular strains and evolution characteristics of Tobacco mosaic virus(TMV) from tobacco plants in Heilongjiang producing area, three samples from Harbin and Mudanjiang tobacco fields were collected and designated as HEB1, HEB2 and MDJ. Their complete genomic fragments were amplified via RT-PCR. Sequence identities, recombination, phylogenetic relationship and selection pressure were analyzed. Results showed that the full-length genome of isolates HEB1, HEB2 and MDJ were 6395 nucleotides(nt)(GenBank accession numbers MH595919,MH595920 and MH595921). HEB1 and MDJ had the highest identities of 98.9% and 99.6%, respectively, with isolate Beipiao(HE818412) from Liaoning province, while MDJ had the highest identity of 97.1% with isolate Chuxiong1(HE818417) from Chuxiong of Yunnan province. HEB1 was a recombinant of isoaltes MDJ and HEB2. In the phylogenetic tree constructed with the complete genomic sequence, TMV was divided into three groups, among which HEB1 and MDJ belong to group Ⅰ(U1) while HEB2 belong to group Ⅱ(Rakkyo). All four proteins of TMV were under negative selection, with CP gene under the highest selection pressure.
引文
[1]Zimmern D.The 5'end group of tobacco mosaic virus RNA is m7G5'ppp5'Gp.[J].Nucleic Acids Research,1975,2(7):1189.
[2]Gallie D R,Feder J N,Schimke R T,et al.Functional analysis of the tobacco mosaic virus tRNA-like structure in cytoplasmic gene regulation.[J].Nucleic Acids Research,1991,19(18):5031-6.
[3]Goelet P,Lomonossoff G P,Butler P J,et al.Nucleotide sequence of tobacco mosaic virus RNA.[J].Proceedings of the National Academy of Sciences of the United States of America,1982,79(19):5818-5822.
[4]谢扬军.烟草普通花叶病毒株系分化研究[M].中国农业科学院硕士研究生论文,2009.XIE Yangjun.The research of TMV isolates differentiation[M].Master Degree thesis of Chinese Academy of Agricultural Sciences,2009.
[5]杨恭,刘相国,邱并生.烟草花叶病毒(普通株中国分离物)及其弱毒疫苗-N14(番茄株)基因组侵染性cDNA核苷酸全序列测定与分析[J].生物工程学报,2000,16(4):437-442.YANG Gong,LIU Xiangguo,QIU Bingsheng.Complete nucleotide sequences and genome structure of two Chinese tobacco mosaic virus isolates deduced from full-length infectious cDNA clones[J].Chinese Journal of Biotechnology,2000,16(4):437-442.
[6]邵碧英,吴祖建,林奇英,等.烟草花叶病毒及其弱毒株基因组的cDNA克隆和序列分析[J].植物病理学报,2003,33(4):296-301.SHAO Biying,WU Zujian,LIN Qiying,et al.Genomic cloning and nucleotide sequence analysis of the cDNAs of Tobacco mosaic virus and its mild strains[J].Acta Phytopathologica Sinica,2003,33(4):296-301.
[7]庞小静,赵慧琪,王德富,等.烟草花叶病毒山西番茄分离物的全基因组序列测定及分析[J].中国蔬菜,2017,1(6):50-55.PANG Xiaojing,ZHAO Huiqi,WANG Defu,et al.Complete Whole Genome Sequence Measure and Analysis of Tobacco mosaic virus Infecting Tomato in Shanxi[J].China Vegetables,2017,1(6):50-55.
[8]Scottolavino E,Du G,Frohman M A.5'end cDNA amplification using classic RACE.[J].Nature Protocols,2006,1(6):2555-2562.
[9]谢联辉,林奇英,谢莉妍,等.福建烟草病毒种群及其发生频率的研究[J].中国烟草学报,1994,2(1):25-32.XIE Lianhui,LIN Qiying,XIE Liyan,et al.Studies on the population and occurrence frequency of tobacco virus diseases in Fujian[J].Acta Tabacaria Sinica,1994,2(1):25-32.
[10]王劲波,王凤龙,钱玉梅,等.山东烟区主要病毒的株系鉴定[J].中国烟草学报,1998,4(1):24-32.WANG Jinbo,WANG Fenglong,QIAN Yvmei,et al.Identification of main viruses in Shandong tobacco region[J].Acta Tabacaria Sinica,1998,4(1):24-32.
[11]Holmes F O.Inheritance of resistance to tobacco-mosaic disease in tobacco[J].Phytopathology,1938,28(8):553-561.
[12]Korbelin J,Willingmann P,Adam G,et al.The complete sequence of tobacco mosaic virus isolate Ohio V reveals a high accumulation of silent mutations in all open reading frames[J].Archives of Virology,2012,157(2):387-389.
[13]丁铭,方琦,张丽珍,等.云南五个不同地区烟草花叶病毒外壳蛋白基因的序列比较[J].中国病毒学,2004,19(1):76-78.DING Ming,FANG Qi,ZHANG Lizhen,et al.Comparision of the coat protein gene seqeunce of tobacco mosaic virus isolated from different regions in Yunnan Province[J].Virologica Sinica,2004,19(1):76-78.
[14]秦西云,卢训,方琦,等.云南烟草中烟草花叶病毒株系比较与分析[J].中国烟草学报,2013(6):106-113.QIN Xiyun,LU Xun,FANG Qi,et al.Comparison of tobacco mosaic virus strains isolated from tobacco grown in different areas in Yunnan Province[J].Acta Tabacaria Sinica,2004,19(1):76-78.
[15]王莉爽,吴石平,杨学辉,等.贵州烟草花叶病毒株系初探[J].贵州农业科学,2012,40(12):88-91.WANG Lishuang,WU Shiping.YANG Xuehui,et al.Preliminary Study on Tobacco mosaic virus strains in Guizhou province[J].Guizhou Agricultural Sciences,2012,40(12):88-91.
[16]刘天波,周志成,彭曙光,等.基于全基因组编码区序列的烟草花叶病毒分子进化分析[J].植物保护,2017,43(5):87-92,107.LIU Tianbo,ZHOU Zhicheng,PENG Shuguang,et al.Complete genomic coding sequence-based molecular evolutionary analysis of Tobacco mosaic virus[J].Plant Protection,2017,43(5):87-92,107.
[17]刘金亮,王凤婷,魏毅,等.烟草花叶病毒南瓜分离物CP基因的克隆、序列分析及其原核表达[J].华北农学报,2010,25(5):6-10.LIU Jinliang,WANG Fengting,WEI Yi,et al.Cloning,Sequence analysis and prokaryotic expression of the coat protein gene of Tobacco mosaic virus from Pumpkin Plant[J].Acta Agriculturae Boreali-Sinica,2010,25(5):6-10.
[18]黄金光,邓丛良,范在丰,等.烟草花叶病毒丁香分离物的分离与鉴定[J].植物病理学报,2004,34(3):215-220.HUANG Jinguang,DENG Congliang,FAN Zaifeng,et al.Isolation and Identification of Tobacco mosaic virus infecting Syringa oblate[J].Acta Phytopathologica Sinica,2004,34(3):215-220.
[19]Worobey M,Holmes E C.Homologous Recombination in GBVirus C/Hepatitis G Virus[J].Molecular Biology&Evolution,2001,18(2):254-61.
[20]Walt E V D,Rybicki E P,Varsani A,et al.Rapid host adaptation by extensive recombination[J].Journal of General Virology,2009,90(3):734-746.
[21]Yin X,Zheng F Q,Tang W,et al.Genetic structure of rice blackstreaked dwarf virus populations in China[J].Archives of Virology,2013,158(12):2505-2515.
[22]Zhang C L,Gao R,Wang J,et al.Molecular variability of Tobacco vein banding mosaic virus populations[J].Virus Research,2011,158(1/2):188.
[2]Gallie D R,Feder J N,Schimke R T,et al.Functional analysis of the tobacco mosaic virus tRNA-like structure in cytoplasmic gene regulation.[J].Nucleic Acids Research,1991,19(18):5031-6.
[3]Goelet P,Lomonossoff G P,Butler P J,et al.Nucleotide sequence of tobacco mosaic virus RNA.[J].Proceedings of the National Academy of Sciences of the United States of America,1982,79(19):5818-5822.
[4]谢扬军.烟草普通花叶病毒株系分化研究[M].中国农业科学院硕士研究生论文,2009.XIE Yangjun.The research of TMV isolates differentiation[M].Master Degree thesis of Chinese Academy of Agricultural Sciences,2009.
[5]杨恭,刘相国,邱并生.烟草花叶病毒(普通株中国分离物)及其弱毒疫苗-N14(番茄株)基因组侵染性cDNA核苷酸全序列测定与分析[J].生物工程学报,2000,16(4):437-442.YANG Gong,LIU Xiangguo,QIU Bingsheng.Complete nucleotide sequences and genome structure of two Chinese tobacco mosaic virus isolates deduced from full-length infectious cDNA clones[J].Chinese Journal of Biotechnology,2000,16(4):437-442.
[6]邵碧英,吴祖建,林奇英,等.烟草花叶病毒及其弱毒株基因组的cDNA克隆和序列分析[J].植物病理学报,2003,33(4):296-301.SHAO Biying,WU Zujian,LIN Qiying,et al.Genomic cloning and nucleotide sequence analysis of the cDNAs of Tobacco mosaic virus and its mild strains[J].Acta Phytopathologica Sinica,2003,33(4):296-301.
[7]庞小静,赵慧琪,王德富,等.烟草花叶病毒山西番茄分离物的全基因组序列测定及分析[J].中国蔬菜,2017,1(6):50-55.PANG Xiaojing,ZHAO Huiqi,WANG Defu,et al.Complete Whole Genome Sequence Measure and Analysis of Tobacco mosaic virus Infecting Tomato in Shanxi[J].China Vegetables,2017,1(6):50-55.
[8]Scottolavino E,Du G,Frohman M A.5'end cDNA amplification using classic RACE.[J].Nature Protocols,2006,1(6):2555-2562.
[9]谢联辉,林奇英,谢莉妍,等.福建烟草病毒种群及其发生频率的研究[J].中国烟草学报,1994,2(1):25-32.XIE Lianhui,LIN Qiying,XIE Liyan,et al.Studies on the population and occurrence frequency of tobacco virus diseases in Fujian[J].Acta Tabacaria Sinica,1994,2(1):25-32.
[10]王劲波,王凤龙,钱玉梅,等.山东烟区主要病毒的株系鉴定[J].中国烟草学报,1998,4(1):24-32.WANG Jinbo,WANG Fenglong,QIAN Yvmei,et al.Identification of main viruses in Shandong tobacco region[J].Acta Tabacaria Sinica,1998,4(1):24-32.
[11]Holmes F O.Inheritance of resistance to tobacco-mosaic disease in tobacco[J].Phytopathology,1938,28(8):553-561.
[12]Korbelin J,Willingmann P,Adam G,et al.The complete sequence of tobacco mosaic virus isolate Ohio V reveals a high accumulation of silent mutations in all open reading frames[J].Archives of Virology,2012,157(2):387-389.
[13]丁铭,方琦,张丽珍,等.云南五个不同地区烟草花叶病毒外壳蛋白基因的序列比较[J].中国病毒学,2004,19(1):76-78.DING Ming,FANG Qi,ZHANG Lizhen,et al.Comparision of the coat protein gene seqeunce of tobacco mosaic virus isolated from different regions in Yunnan Province[J].Virologica Sinica,2004,19(1):76-78.
[14]秦西云,卢训,方琦,等.云南烟草中烟草花叶病毒株系比较与分析[J].中国烟草学报,2013(6):106-113.QIN Xiyun,LU Xun,FANG Qi,et al.Comparison of tobacco mosaic virus strains isolated from tobacco grown in different areas in Yunnan Province[J].Acta Tabacaria Sinica,2004,19(1):76-78.
[15]王莉爽,吴石平,杨学辉,等.贵州烟草花叶病毒株系初探[J].贵州农业科学,2012,40(12):88-91.WANG Lishuang,WU Shiping.YANG Xuehui,et al.Preliminary Study on Tobacco mosaic virus strains in Guizhou province[J].Guizhou Agricultural Sciences,2012,40(12):88-91.
[16]刘天波,周志成,彭曙光,等.基于全基因组编码区序列的烟草花叶病毒分子进化分析[J].植物保护,2017,43(5):87-92,107.LIU Tianbo,ZHOU Zhicheng,PENG Shuguang,et al.Complete genomic coding sequence-based molecular evolutionary analysis of Tobacco mosaic virus[J].Plant Protection,2017,43(5):87-92,107.
[17]刘金亮,王凤婷,魏毅,等.烟草花叶病毒南瓜分离物CP基因的克隆、序列分析及其原核表达[J].华北农学报,2010,25(5):6-10.LIU Jinliang,WANG Fengting,WEI Yi,et al.Cloning,Sequence analysis and prokaryotic expression of the coat protein gene of Tobacco mosaic virus from Pumpkin Plant[J].Acta Agriculturae Boreali-Sinica,2010,25(5):6-10.
[18]黄金光,邓丛良,范在丰,等.烟草花叶病毒丁香分离物的分离与鉴定[J].植物病理学报,2004,34(3):215-220.HUANG Jinguang,DENG Congliang,FAN Zaifeng,et al.Isolation and Identification of Tobacco mosaic virus infecting Syringa oblate[J].Acta Phytopathologica Sinica,2004,34(3):215-220.
[19]Worobey M,Holmes E C.Homologous Recombination in GBVirus C/Hepatitis G Virus[J].Molecular Biology&Evolution,2001,18(2):254-61.
[20]Walt E V D,Rybicki E P,Varsani A,et al.Rapid host adaptation by extensive recombination[J].Journal of General Virology,2009,90(3):734-746.
[21]Yin X,Zheng F Q,Tang W,et al.Genetic structure of rice blackstreaked dwarf virus populations in China[J].Archives of Virology,2013,158(12):2505-2515.
[22]Zhang C L,Gao R,Wang J,et al.Molecular variability of Tobacco vein banding mosaic virus populations[J].Virus Research,2011,158(1/2):188.