ToCV和TYLCV复合侵染番茄后部分病毒基因序列分析及实时荧光定量PCR分析

doi:10.7668/hbnxb.201751035

摘要/Abstract

摘要： 番茄褪绿病毒病（ToCV）和番茄黄化曲叶病毒（TYLCV）存在复合侵染的现象，其发病率逐年递增，成为一种新的威胁番茄产业的病毒病害。为了研究复合侵染样品中2种病毒的互作和基因变异情况，对天津番茄产区发生的TYLCV全基因组和ToCV的CP基因和HSP基因进行了克隆和序列分析。结果表明，与单独侵染样品进行比对，TYLCV的基因序列在复合侵染的样品中共有6处碱基发生了变异，分别为73位A→C、92位A→G、347位C→T、1 209位C→T、1 618位A→G、2 107位A→T，除73位和92位的变异未在编码区，其余的变异碱基均在ORF框内，其中1 209位为同义突变，其他均发生了错义突变。ToCV的CP基因有1处同义突变，ToCV的HSP基因共有4处碱基变异，其中826位由T→C和1 166位由G→A均为同义突变，1 395位和1 630位均由A→G，为错义突变。利用实时荧光定量PCR技术对单独侵染和复合侵染的番茄样品中TYLCV和ToCV病毒积累量进行分析。结果表明，在田间单独侵染和复合侵染的样品中，TYLCV的拷贝数差异不显著，ToCV的拷贝数在单独侵染和复合侵染的样品中差异也不显著。在复合侵染的样品中，TYLCV的拷贝数约为ToCV的262~436倍。

关键词: 番茄黄化曲叶病毒, 番茄褪绿病毒, 复合侵染, 序列分析, 实时荧光定量PCR

Abstract: There exists the compound infection of Tomato chlorosis virus (ToCV) and Tomato yellow leaf curl virus (TYLCV), and the incidence of the two diseases presents a tread of increase in recent years, which becomes a new threat to tomato farming. In order to study the interaction and variation of the two virus genes after single and mixed infection, the molecular identification and sequence analysis of the CP gene and the HSP gene of ToCV and the gene of TYLCV occurred in Tianjin were carried out. The results showed that, compared with the single infection samples, 6 base mutations of TYLCV were found in mixed infection samples, i.e. 73rd A→C, 92nd A→G, 347th C→T, 1 209th C→T, 1 618th A→G and 2 107th A→T, among which the 73rd and 92nd mutations were not in the coding region and the other four were located in the ORF box with 3 missense mutations and one samesense mutation(1 209th). There was also one samesense mutation in the CP gene of ToCV. There were 4 base mutations in HSP gene of ToCV, among which the 826th T→C and 1 166th G→A were samesense mutations, while the 1 395th A→G and 1 630th A→G were missense mutations. The accumulation amount of TYLCV and ToCV in tomato samples after single infection and mixed infection were analyzed by RT-qPCR. The results showed that the accumulations of TYLCV and ToCV had no significant difference between single infection and mixed infection. In the mixed infection samples, the copy of TYLCV was about 262-436 times that of ToCV.

Key words: TYLCV, ToCV, Mixed infection, Sequence analysis, Real-time quantitative PCR

中图分类号:

宋建, 薛俊, 金凤媚, 孙海波, 张越, 王姝, 范寰, 周祥明, 陈锐. ToCV和TYLCV复合侵染番茄后部分病毒基因序列分析及实时荧光定量PCR分析[J]. 华北农学报, 2019, 34(2): 95-102. doi: 10.7668/hbnxb.201751035.

SONG Jian, XUE Jun, JIN Fengmei, SUN Haibo, ZHANG Yue, WANG Shu, FAN Huan, ZHOU Xiangming, CHEN Rui. Partial Virus Gene Sequence and Real-time Quantitative PCR Analysis of Tomato after Mixed Infection of ToCV and TYLCV[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2019, 34(2): 95-102. doi: 10.7668/hbnxb.201751035.

http://www.hbnxb.net/CN/Y2019/V34/I2/95

参考文献

[1] Pasev G,Radeva V,Kostova D. First report of Tomato infectious chlorosis virus on tomato in Bulgaria[J]. Journal of Phytopathology,2012,160(3):160-162.doi:10.1111/j.1439-0434.2011.01874.
[2] Salem N M,Mansour A N,Abdeen A O,Araj S,Khrfan W I. First report of tomato chlorosis virus infecting tomato crops in Jordan[J]. Plant Disease,2015,99(9):1503.doi:10.1094/PDIS-01-15-0130-PDN.
[3] 周涛,杨普云,赵汝娜,师迎春,原锴,范在丰.警惕番茄褪绿病毒在我国的传播和危害[J].植物保护,2014,40(5):196-199.doi:10.3969/j.issn.0529-1542.2014.05.038.
Zhou T,Yang P Y,Zhao R N,Shi Y C,Yuan K,Fan Z F.Alarming the transmission and damage of Tomato chlorosis virus in China[J].Plant Protection,2014,40(5):196-199.
[4] 刘永光,魏家鹏,乔宁,李美芹,刘晓明,竺晓平.番茄褪绿病毒在山东暴发及其防治措施[J].中国蔬菜,2014(5):67-69.doi:10.3969/j.issn.1000-6346.2014.05.018.
Liu Y G,Wei J P,Qiao N,Li M Q,Liu X M,Zhu X P. Outbreak of Tomato chlorosis virus in Shandong and its control measures[J].China Vegetables,2014(5):67-69.
[5] 汤亚飞,何自福,佘小漫,蓝国兵.侵染广东番茄的番茄褪绿病毒分子鉴定[J].植物保护,2017,43(2):133-137.doi:10.3969/j.issn.0529-1542.2017.02.022.
Tang Y F,He Z F,She X M,Lan G B. Molecular identification of Tomato chlorosis virus infecting tomato in Guangdong province[J]. Plant Protection,2017,43(2):133-137.
[6] 王翠琳,冯佳,孙晓辉,王少立,赵静,刘金亮,竺晓平.北方四省区番茄褪绿病毒的分子鉴定[J].植物保护,2017,43(2):141-145.doi:10.3969/j.issn.0529-1542.2017.02.024.
Wang C L,Feng J,Sun X H,Wang S L,Zhao J,Liu J L,Zhu X P. Molecular identification of Tomato chlorosis virus from four provinces or autonomous region in northern China[J].Plant Protection,2017,43(2):141-145.
[7] 金凤媚,薛俊,宋建.天津地区番茄褪绿病毒的分子检测与基因组部分序列分析[J].华北农学报,2016,31(2):23-27.doi:10.7668/hbnxb.2016.02.005.
Jin F M,Xue J,Song J. Molecular detection and partial genome sequence analysis of tomato chlorosis virus in Tianjin[J]. Acta Agriculturae Boreali-Sinica,2016,31(2):23-27.
[8] 金凤媚,薛俊,郏艳红,周祥明,王建江,刘仲齐.天津地区番茄黄化曲叶病毒DNA-A的克隆和序列分析[J]. 华北农学报,2011,26(1):58-62.doi:10.7668/hbnxb.2011.01.013.
Jin F M,Xue J,Jia Y H,Zhou X M,Wang J J,Liu Z Q. Cloning and sequence analysis of TYLCV DNA-A in Tianjin[J].Acta Agriculturae Boreali-Sinica,2011,26(1):58-62.
[9] Chinnaraja C,Ramkissoon A,Ramsubhag A,Jayaraj J. First report of tomato yellow leaf curl virus Infecting tomatoes in Trinidad[J].Plant Disease,2016,100(9):1958.doi:10.1094/PDIS-04-16-0446-PDN.
[10] Verdin E,Desbiez C,Catherine W S,Gognalons P,Ahmed K P, Gronenborn B,Mirzayeva S,Sultanova N,Mammadov A,Huseynova I. First report of tomato yellow leaf curl virus infecting tomato in Azerbaijan[J]. Journal Plant Pathology,2018,100(2):335.doi:10.1007/s42161-018-0050-x.
[11] 张前荣,李大忠,朱海生,刘建汀,李永平,温庆放.番茄黄化曲叶病毒研究进展[J].分子植物育种,2017,15(9):3709-3716.
Zhang Q R,Li D Z,Zhu H S,Liu J T,Li Y P,Wen Q F.A review of tomato yellow leaf curl virus[J].Molecular Plant Breeding,2017,15(9):3709-3716.
[12] 宋晰,师迎春,张世晨,梁彦,王廿,陈善义,周涛.北京地区番茄黄化曲叶病病毒分离物测定及株系的初步鉴定[J].植物病理学报,2013,43(2):113-119.doi:10.3969/j.issn.0412-0914.2013.02.001.
Song X,Shi Y C,Zhang S C,Liang Y,Wang N,Chen S Y,Zhou T. Detection and preliminary strain identification of the pathogen induing tomato yellow leaf curl disease in Beijing[J].Acta Phytopathologica Sinica,2013,43(2):113-119.
[13] 曹骞,李晶,买热木古丽·克依木,王惠卿,李国志,马德英.新疆地区烟粉虱生物型的区域分布及其携带的番茄黄化曲叶病毒检测[J].昆虫学报,2013,56(6):652-664.
Cao Q,Li J,Mairemuguli K Y M,Wang H J,Li G Z,Ma D Y. Biotype distribution and infection status of tomato yellow leaf curl virus(TYLCV) in Bemisia tabaci(Hemiptera:Aleyrodidae) in Xinjiang, northwestern China[J]. Acta Entomalogica Sinica,2013,56(6):652-664.
[14] Rabie M,Ratti C,Aleem E A,Fattouh F. Detection and molecular characterization of tomato yellow leaf curl virus naturally infecting Lycopersicon esculentum in Egypt[J]. Actavirologica,2017,61(3):252-263.doi:10.4149/av_2017_303.
[15] Ebtisam A,Hanadi A,Ahmed B H,Aqeel H AI. First report of tomato yellow leaf curl virus infecting cucumber in Kuwait[J]. Plant Disease,2016,100(3):656.doi:10.1094/PDIS-07-15-0830-PDN.
[16] Alabi O J,Rwahnih A M.,Brown J K,Idris A M,Gregg L,Kmieciak E,Setamou M,Jifon J L. First report of papaya (Carica papaya) naturally infected with the introduced tomato yellow leaf curl virus-Israel[J]. Plant Disease,2016,100(9):1959.doi:10.1094/PDIS-04-16-0469-PDN.
[17] Zhou Y,Luo C,Zhao J,Wei S J,Chen Z,Yan J Y,Li X H. First report of tomato yellow leaf curl virus in viola prionantha in China[J]. Plant Disease,2016,100(1):231.doi:10.1094/PDIS-01-15-0104-PDN.
[18] Garcia-cano E,Resende R O,Fernandez-Munoz R,Moriones E. Synergistic interaction between Tomato chlorosis virus and Tomato spotted wilt virus results in breakdown of resistance in tomato[J].Phytopathology,2006,96(11):1263-1269.doi:10.1094/phyto-96-1263.
[19] Alfaro-Fernández A,Medina V,Cordoba M C,Font M I,Jornet J,Cebrian M C,Jorda C. Ultrastructrual aspects of tomato leaves infected by Tomato torradovirus(ToTV)and co-infected by other viruses[J]. Plant Pathology,2010,59(2):231-239.doi:10.1111/j.1365-3059.2009.02215.
[20] Martínez-Zubiaur Y, FialloOlivé E,Tripp J C,Bustamante R R. First report of Tomato chlorosis virus infecting tomato in single and mixed infections with Tomato yellow leaf curl virus in Cuba[J]. Plant Disease,2008,92(5):836.doi:10.1094/PDIS-92-5-0836C.
[21] 赵黎明,李刚,刘永光,国家进,魏家鹏,竺晓平.山东设施番茄上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.doi:10.3969/j.issn.1000-6346.2014.12.006.
Zhao L M,Li G,Liu Y G,Guo J J,Wei J P,Zhu X P. Molecular Identification on Mixed Infections of Tomato chlorosis virus and Tomato yellow leaf curl virus[J].China Vegetables,2014(12):15-20.
[22] 吴淑华,李廷芳,赵文浩,程兆榜,郭青云, 赵统敏, 余文贵, 朱叶芹, 季英华.江苏省番茄黄化曲叶病毒和褪绿病毒复合侵染的分子检测[J].园艺学报,2016,43(1):89-99.doi:10.16420/j.issn.0513-353x.2015-0584.
Wu S H,Li T F,Zhao W H,Cheng Z B,Guo Q Y,Zhao T M,Yu W G,Zhu Y Q,Ji Y H. Molecular identification on mixed infection of tomato yellow leaf curl virus and tomato chlorosis virus on tomato in Jiangsu Province[J].Acta Horticulturae Sinica,2016,43(1):89-99.
[23] 刘微,史晓斌,唐鑫,张宇,张德咏,周序国,刘勇.云南番茄褪绿病毒和番茄黄化曲叶病毒复合侵染的分子鉴定[J].园艺学报,2018,45(3):552-560.doi:10.16420/j.issn.0513-353x.2017-0735.
Liu W,Shi X B,Tang X,Zhang Y,Zhang D Y,Zhou X G,Liu Y. Molecular Identification of Tomato chlorosis virus and Tomato yellow leaf curl virus in Yunnan Province[J].Acta Horticulturae Sinica,2018,45(3):552-560.
[24] Harrison B D,Swanson M M,Fargette D. Begomovirus coat protein:serology,variation and functions[J]. Physiological and Molecular Plant Pathology,2002,60:257-271.doi:10.1006/pmpp.2002.0404.
[25] Laufs J,Jupin I,David C,Schumacher S, Heyraud-Nitschke F, Gronenborn B. Geminivirus replication:Genetic and biochemical characterization of Rep protein function[J]. A Review Biochimie, 1995,77:765-773.doi:10.1177/14.12.884.
[26] Sunter G,Bisaro D M. Transactivation of Geminivirus AR1 and BR1 gene expression by the viral AL2 gene product occurs at the level of transcription[J]. Plant Cell,1992,4,1321-1331.doi:10.1105/tpc.4.10.1321.
[27] Harrison B D,Zhou X,Otim-Nape G W. Role of a novel type of double infection in Geminivirus-induced epidemic of severe cassava mosaic in Uganda[J]. Annals of Applied Biology,1997,131:437-448.doi:10.1111/j.1744-7348.1997.tb05171.
[28] Vance V B. Replication of potato virus×RNA is altered in coinfections with Potato virus Y[J].Virology,1991,182(9):486-494.doi:10.1016/0042-6822(91)90589-4.
[29] Xia Z H,Zhao Z X, Chen,Li M J,Zhou T,Deng C L,Zhou Q, Fan Z F. Synergistic infection of two viruses MCMV and SCMV increases the accumulations of both CMV and MCMV-derived siRNAs in maize[J]. Scientific Reports,2016,6:20520.doi:10.1038/srep20520.
[30] Garcia-cano E,Resende R O, Fernandez-Munoz R,Moriones E. Synergistic interaction between Tomato chlorosis virus and Tomato spotted wilt virus results in breakdown of resistance in tomato[J].Phytopathology,2006,96(11):1263-1269.doi:10.1094/phyto-96-1263.
[31] Valkonen J. Accumulation of potato virus Y is enhanced in Solatiumbrevidens also infected with tobacco mosaic virus of potato spindle tuber viroid[J].Annals of Applied Biology,1992,121(2):321-32.doi:10.1111/j.1744-7348.1992.tb03445.
[32] Alabi O J,Rwahnih M A,Jifon J L,Sétamou M,Brown J K, Gregg L, Park J W.Mixed infection of Lettuce chlorosis virus, Papaya ringspot virus, and Tomato yellow leaf curl virus-IL detected in a Texas Papaya Orchard affected by a virus-Like disease outbreak[J]. Plant Disease,2017,101(7):1094-1102.
[33] 鲁瑞芳,李为民,彭学贤.植物病毒协生作用及其分子机理[J].中国病毒学,2001,16(3):195-201.
Lu R F,Li W M,Peng X X. Molecular mechanism of synergism of plant virus[J].Virological Sinica,2001,16(3):195-201.
[34] 郭兴启,冯炘,李向东,郭恒俊,李照会.PVY/PVX协生作用对病毒浓度及寄主细胞超微结构的影响[J].中国农业科学,2003,36(3):281-286.doi:10.3321/j.issn:0578-1752.2003.03.009.
Guo X Q,Feng X,Li X D,Guo H J,Li Z H.Effect of synergism between PVY and PVX on the viruses titer and the ultrastructure of infected cell of tobacco plant[J].Scientia Agricultura Sinica,2003,36(3):281-286.

选择文件类型/文献管理软件名称

选择包含的内容