基于小RNA深度测序的烟草脉坏死病病原分子鉴定及其全基因组序列分析  

Molecular identification of tobacco vein necrosis pathogen and complete genome sequence analysis based on small RNA deep sequencing

作　　者：莫翠萍 陈锦清[1] 韦学平 崔丽贤[1] 罗婉笛 李金哲 谢慧婷[1] 秦碧霞[1] 蔡健和[1] 李战彪[1] MO Cui-ping;CHEN Jin-qing;WEI Xue-ping;CUI Li-xian;LUOWan-di;LI Jin-zhe;XIE Hui-ting;QIN Bi-xia;CAI Jian-he;LI Zhan-biao(Plant Protection Research Institute,Guangxi Academy of Agricultural Science/Key Laboratory of Green Prevention and Control on Fruits and Vegetables in South China,Ministry of Agriculture and Rural Affairs/Guangxi Key Laboratory of Biology for Crop Diseases and Insect Pests,Nanning,Guangxi 530007,China;Baise Tobacco Company of Guangxi,Baise,Guangxi 533000,China;College of Agriculture,Yangtze University,Jingzhou,Hubei 434000,China)

机构地区：[1]广西农业科学院植物保护研究所/农业农村部华南果蔬绿色防控重点实验室/广西作物病虫害生物学重点实验室,广西南宁530007 [2]广西烟草公司百色市公司,广西百色533000 [3]长江大学农学院,湖北荆州434000

出　　处：《南方农业学报》2023年第5期1387-1396,共10页Journal of Southern Agriculture

基　　金：广西自然科学基金项目(2020GXNSFAA259003);广西农业科学院基本科研业务专项(桂农科2020YM92,桂农科2021YT071)。

摘　　要：【目的】明确引起广西河池市南丹县烟草呈现斑驳花叶、叶脉坏死等症状的病毒病原,为烟草病毒病的综合防治提供理论依据。【方法】2022年5月,从河池市南丹县采集5份表现叶脉坏死、花叶等症状的烟草叶片样品,采用小RNA深度测序技术、反转录PCR(RT-PCR)、摩擦接种、分段克隆、系统进化及重组分析等方法对样品进行鉴定及遗传进化分析。【结果】小RNA深度测序获得与GenBank数据库中马铃薯Y病毒(Potato virus Y,PVY)不同分离物具有较高核苷酸相似性(99.00%~100.00%)的5条序列,将5条序列拼接后获得1条全长为9708 nt的全基因组序列;通过鉴别寄主苋色藜(Chenopodium amaranticolor)单斑分离的方法获得单一病毒,利用该分离纯化后的病毒单斑接种普通烟K326和本氏烟,其中K326的新叶产生坏死症状,而本氏烟的新叶呈不规则深绿色病斑症状;分别提取K326和本氏烟的叶片样品总RNA,通过RT-PCR检测、分段克隆的方法获得病毒分离物的全基因组序列,结果显示2个烟草品种中的病毒序列与小RNA深度测序获得的序列相似性达99.90%;将序列在GenBank中进行BLAST分析发现,研究获得的序列与已登录GenBank的PVY各分离物具有较高的核苷酸相似性,其中与我国黑龙江省马铃薯分离物PVY HLJ26(MF134425)的核苷酸相似性最高,为99.01%,表明研究获得的病毒序列为PVY分离物,命名为PVY-GXnd1(OP131591)。重组分析发现,PVY-GXnd1是由PVYO-139(U09509.1)、PVYN-Mont(AY884983.1)和PVYN-605(X97895.1)重组而来的新PVY分离物,重组主要发生在3个区域,分别是1~498、2415~5816和8555~9373 nt,覆盖PVY的P1、P3、6K1、CI、6K2、Vpg、Nib和CP蛋白编码区,包含了PVYN-Wi和PVYNTN株系的2种不同重组结构特征,表明PVYGXnd1株系更接近PVYNTN-NW株系;基于PVY编码区构建的系统发育进化树分析表明,PVY-GXnd1与我国黑龙江省分离物PVY HLJ26处于同一小分支,具有较近的亲缘关系,而该分�【Objective】The purpose of the study was to clarify the virus pathogens causing tobacco mosaic,vein ne-crosis and other symptoms in Nandan County,Hechi City,Guangxi,so as to provide theoretical basis for the comprehen-sive prevention and control of tobacco virus diseases.【Method】In May 2022,five tobacco leaf samples showing symp-toms such as vein necrosis and mosaic were collected from Nandan,Hechi.The samples were identified and analyzed for genetic evolution by using small RNA deep sequencing,reverse transcription polymerase chain reaction(RT-PCR),fric-tion inoculation,segmented cloning,phylogenetic evolution and recombination analysis.【Result】Small RNA sequencing obtained five sequences with high nucleotide identity(99.00%-100.00%)with different potato virus Y(PVY)isolates in the GenBank database.Five sequences were assembled to obtain a complete genome sequence with a total length of 9708 nt.The single virus was obtained by identifying a single spot isolate of the host Chenopodium amaranticolor.The purified virus single spot was used to inoculate tobacco variety K326 and Nicotiana benthamiana,in which the new leaves of K326 produced necrotic symptoms,while the new leaves of N.benthamiana showed irregular dark green spot symptoms.Total RNA was extracted from the leaf samples of K326 and N.benthamiana,respectively,and the whole genome se-quences of the virus isolates were obtained by RT-PCR detection and segmented cloning.The results showed that the iden-tity of the virus sequences of the two tobacco varieties with the sequences obtained by small RNA deep sequencing was 99.90%.BLAST analysis of the sequences in GenBank showed that the sequences obtained in the study had high nucleo-tide identity with the PVY isolates that had been published in GenBank,and the highest nucleotide identity was 99.01%with the isolate from Heilongjiang Province,China,PVY HLJ26(MF134425).It indicated that the virus sequence ob-tained from the study was one isolate of PVY,and named as PVY-GXnd1(OP131591).The results of reco

关 键 词：烟草 小RNA深度测序 马铃薯Y病毒 重组 叶脉坏死 广西 

分 类 号：S435.72[农业科学—农业昆虫与害虫防治]