新德里番茄曲叶病毒西瓜分离物的全基因组序列测定及分析

doi:10.3785/j.issn.1008-9209.2023.05.122

浙江大学学报（农业与生命科学版）

2023, Vol. 49

Issue (5): 687-695 DOI: 10.3785/j.issn.1008-9209.2023.05.122

作物重要细菌和病毒病害专题

新德里番茄曲叶病毒西瓜分离物的全基因组序列测定及分析

程雨欣(

),胡红霞,梁燕青,钱亚娟(

)

浙江大学农业与生物技术学院生物技术研究所，浙江杭州 310058

Whole genome sequencing and analysis of isolates of tomato leaf curl New Delhi virus collected from watermelon

Yuxin CHENG(

),Hongxia HU,Yanqing LIANG,Yajuan QIAN(

)

Institute of Biotechnology, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, Zhejiang, China

全文: PDF(3557 KB) HTML

摘要：

从上海采集到表现卷叶和黄化症状的西瓜病叶，为明确其病原类型，本研究提取病叶RNA并进行小RNA高通量测序，经序列拼接比对和斑点酶联免疫吸附试验（dot-enzyme linked immunosorbent assay, Dot-ELISA），证实病样感染新德里番茄曲叶病毒（tomato leaf curl New Delhi virus, ToLCNDV）。进一步通过滚环复制（rolling circle replication, RCR）富集DNA，利用背靠背引物进行聚合酶链反应（polymerase chain reaction, PCR）扩增，获得2个ToLCNDV西瓜分离物的DNA-A和DNA-B全长序列，暂命名为ToLCNDV SH-WM1和ToLCNDV SH-WM2。使用MEGA 11.0、SDT v1.2等软件进行系统进化分析，结果表明，ToLCNDV SH-WM1与ToLCNDV SH-WM2及已报道的ToLCNDV其他中国分离物的亲缘关系较近，其全核苷酸序列相似度为98.99%～99.70%。全基因组多态性分析及群体变异分析显示，ToLCNDV中国分离物的单核苷酸变异率小于3%，其中，同义突变位点46个，非同义突变位点29个，不存在插入或缺失突变，各个编码蛋白中AC4蛋白氨基酸序列最为保守。本研究揭示ToLCNDV中国分离物具有较低的种内遗传变异。

关键词： 新德里番茄曲叶病毒; 西瓜; 基因组变异

Abstract:

Watermelon diseased leaves exhibiting leaf curl and yellowing symptoms were collected from Shanghai. To clarify the pathogenic type, small RNA high-throughput sequencing of the diseased leaves was performed, and the diseased samples were confirmed to be infected by tomato leaf curl New Delhi virus (ToLCNDV) through sequence splicing and dot-enzyme linked immunosorbent assay (Dot-ELISA). The DNA was further enriched by rolling circle replication (RCR) and was amplified by polymerase chain reaction (PCR) using back-to-back primers to obtain the full-length DNA-A and DNA-B sequences of two ToLCNDV watermelon isolates, which were tentatively named as ToLCNDV SH-WM1 and ToLCNDV SH-WM2. Phylogenetic analysis using MEGA 11.0 and SDT v1.2 softwares showed that ToLCNDV SH-WM1 was closely related to ToLCNDV SH-WM2 and other reported ToLCNDV isolates from China, with a total nucleotide sequence similarity of 98.99%-99.70%. Whole genome polymorphism analysis and population variation analysis showed that the single nucleotide variation rate of ToLCNDV isolates from China was less than 3%, with a total of 46 synonymous mutation sites and 29 non-synonymous mutation sites. There were no insertion or deletion mutations, and the amino acid sequence of AC4 protein was the most conservative among all encoded proteins. This study reveals that the ToLCNDV isolates from China have low intraspecific genetic variation.

Key words: tomato leaf curl New Delhi virus watermelon genomic variation

收稿日期: 2023-05-12 出版日期: 2023-11-03

CLC:

Q939.46

基金资助: 国家自然科学基金项目(32272480)

通讯作者: 钱亚娟 E-mail: 22016078@zju.edu.cn;yjqian@zju.edu.cn

作者简介: 程雨欣（https://orcid.org/0009-0000-7230-1062），E-mail：22016078@zju.edu.cn

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引用本文:

程雨欣,胡红霞,梁燕青,钱亚娟. 新德里番茄曲叶病毒西瓜分离物的全基因组序列测定及分析[J]. 浙江大学学报（农业与生命科学版）, 2023, 49(5): 687-695.

Yuxin CHENG,Hongxia HU,Yanqing LIANG,Yajuan QIAN. Whole genome sequencing and analysis of isolates of tomato leaf curl New Delhi virus collected from watermelon. Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(5): 687-695.

链接本文:

https://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2023.05.122 或 https://www.zjujournals.com/agr/CN/Y2023/V49/I5/687

引物名称

Primer name

引物序列（5 $′$ →3 $′$ ）

Primer sequence (5 $′$ →3 $′$ )

SH-WM1 DNA-A

F: GTGAAGAACATGCATCGTGATCGT

R: CGTTGCTGTGCTCGGTTCAT

SH-WM1 DNA-B

F: TCGTGAATGGGGTACACTTCTGT

R: CGTTTGTGTTCTCCACTCGGTAC

SH-WM2 DNA-A

F: GTGAAGAACATGCATCGTGATCGT

R: CGTTGCTGTGCTCGGTTCAT

SH-WM2 DNA-B

F: TCGTGAATGGGGTACACTTCTGT

R: CGTTTGTGTTCTCCACTCGGTAC

表 1 引物序列信息

图1 西瓜病叶样品在田间的典型症状

图2 利用PCR（A）和Dot-ELISA（B）鉴定感染ToLCNDV的西瓜病叶样品M：DNA标志物；WT：野生型。

图 3 ToLCNDV SH-WM的基因组结构

图 4 ToLCNDV分离物DNA-A（A）与DNA-B（B）的进化系统树

ToLCNDV分离物 ToLCNDV isolate	GenBank登录号 GenBank accession No.					核苷酸序列相似度 Nucleotide sequence similarity/%
ToLCNDV分离物 ToLCNDV isolate	DNA-A		DNA-B			DNA-A			DNA-B
ToLCNDV SH-WM2 (watermelon)	OQ957166		OQ957168			99.70			99.62
ToLCNDV 0823-1 (melon)	OQ190939		OQ190949			99.63			99.33
ToLCNDV 0823-2 (melon)	OQ190940		OQ190950			99.63			99.33
ToLCNDV 0923-1 (melon)	OQ190941		OQ190951			99.48			99.44
ToLCNDV 0923-3 (melon)	OQ190942		OQ190952			99.45			99.44
ToLCNDV 7856 (cucumber)	OQ190943		OQ190953			99.45			99.33
ToLCNDV 7922 (luffa)	OQ190948		OQ190958			99.45			99.33
ToLCNDV 8011 (cucumber)	OQ190944		OQ190954			99.41			99.33
ToLCNDV 8006 (cucumber)	OQ190945		OQ190955			99.45			99.33
ToLCNDV 8066 (cucumber)	OQ190946		OQ190956			99.67			99.33
ToLCNDV 8087 (luffa)	OQ190947		OQ190957			99.45			99.33
ToLCNDV Haimen4 (melon)	OP585369		OP683996			99.16			98.99
ToLCNDV Ningbo6 (melon)	OP585370		OP683995			99.26			98.99
ToLCNDV Shanghai1 (melon)	OP683993		OP683994			99.30			98.99
ToLCNDV Zhejiang (tomato)	OP356207		OP356208			99.67			98.99
ToLCNDV分离物 ToLCNDV isolate	氨基酸序列相似度 Amino acid sequence similarity/%
ToLCNDV分离物 ToLCNDV isolate	AC1	AC2		AC3	AC4	AC5	AV1	AV2		BC1	BV1
ToLCNDV SH-WM2 (watermelon)	99.53	100.00		99.27	100.00	100.00	100.00	100.00		99.64	99.75
ToLCNDV 0823-1 (melon)	99.26	99.11		99.51	100.00	99.59	99.87	99.12		98.93	99.75
ToLCNDV 0823-2 (melon)	99.26	99.11		99.51	100.00	99.59	99.87	99.12		98.93	99.75
ToLCNDV 0923-1 (melon)	99.26	99.11		99.51	100.00	99.59	99.61	99.12		98.93	99.75
ToLCNDV 0923-3 (melon)	99.26	99.11		99.51	100.00	99.59	99.61	99.12		98.93	99.75
ToLCNDV 7856 (cucumber)	99.44	99.12		99.51	100.00	99.59	99.74	99.12		98.93	99.75
ToLCNDV 7922 (luffa)	99.44	99.12		99.51	100.00	99.59	99.74	99.12		98.93	99.75
ToLCNDV 8011 (cucumber)	99.17	99.12		99.51	100.00	99.59	99.74	99.12		98.93	99.38
ToLCNDV 8006 (cucumber)	99.17	99.12		99.51	100.00	99.59	99.87	99.12		98.93	99.38
ToLCNDV 8066 (cucumber)	99.17	99.12		99.51	100.00	99.59	99.87	99.12		98.93	99.50
ToLCNDV 8087 (luffa)	99.17	99.12		99.51	100.00	99.59	99.74	99.12		98.93	99.50
ToLCNDV Haimen4 (melon)	99.44	99.41		99.75	100.00	100.00	99.87	99.41		98.93	99.87
ToLCNDV Ningbo6 (melon)	99.44	99.41		99.75	100.00	100.00	99.87	99.41		98.93	99.87
ToLCNDV Shanghai1 (melon)	99.44	99.41		99.75	100.00	100.00	99.87	99.41		98.93	99.87
ToLCNDV Zhejiang (tomato)	99.44	99.41		99.75	100.00	100.00	99.87	99.41		98.93	99.87

表 2 ToLCNDV SH-WM1和其他ToLCNDV中国分离物的核苷酸和氨基酸序列相似性比较

图 5 ToLCNDV西瓜分离物和其他ToLCNDV中国分离物的DNA-A（A）和DNA-B（B）全基因组核苷酸序列相似性比较

图6 ToLCNDV中国分离物DNA-A（A）和DNA-B（B）的种群变异水平

图7 ToLCNDV中国分离物的基因组变异情况分析A. DNA-A；B. DNA-B；C. ORFs。

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