侵染萝卜的油菜花叶病毒广东分离物分子特征及其致病性分析

doi:10.3864/j.issn.0578-1752.2022.14.006

摘要/Abstract

摘要：

【目的】病毒病是萝卜（Raphanus sativus）生产上的主要病害之一,严重危害萝卜生产安全及产业发展。本研究旨在鉴定侵染广东萝卜的病毒种类,探究侵染萝卜的油菜花叶病毒（youcai mosaic virus,YoMV）分子特征及致病性,为萝卜病毒病的防控提供科学依据。【方法】从广东省惠州市萝卜种植基地采集了3个疑似病毒病侵染的萝卜病样及1个无症状样品,提取样品总RNA,利用小RNA深度测序初步探明病毒种类,然后根据测序结果设计病毒特异引物进行RT-PCR验证。应用分段克隆方法扩增病原病毒YoMV基因组1—3 405 nt片段和3 194—6 304 nt片段,然后通过同源重组克隆到pCB301双元载体上,得到YoMV-GD分离物基因组全长序列,同时构建该病毒分离物的侵染性克隆。利用MEGA7软件对YoMV-GD及其他分离物全长核苷酸序列进行系统进化分析。通过侵染性克隆人工注射接种本生烟和普通烟,检验pCB301-YoMV-GD的侵染性,然后再注射接种萝卜和菜心,测定YoMV-GD分离物的致病性。【结果】侵染广东萝卜的病毒包含萝卜隐状病毒3（Raphanus sativus cryptic virus 3,RsCV3）、萝卜黄病毒1（Raphanus sativus chrysovirus 1,RasCV1）、芜菁花叶病毒（turnip mosaic virus,TuMV）、YoMV 4种;YoMV-GD分离物全长序列为6 304 nt,编码4个蛋白,分别为124.7 kD复制相关蛋白、183 kD复制酶、30 kD运动蛋白MP、17.7 kD外壳蛋白CP,与YoMV英国分离物（GenBank登录号：AY318866）核苷酸相似性最高,为99.18%;YoMV-GD分离物与英国、德国及我国上海、重庆分离物的亲缘关系较近;构建的侵染性克隆pCB301-YoMV-GD可以系统侵染本生烟和普通烟,引起严重的坏死、枯斑等症状,具有较强的侵染性;YoMV-GD可以系统侵染萝卜和菜心,引起轻微的黄化和花叶等症状,致病力较弱。【结论】侵染广东萝卜的病毒包含RsCV3、RasCV1、TuMV和YoMV,且以复合侵染形式存在;YoMV遗传进化并没有明显的地域性;YoMV-GD对烟草致病力较强,而对萝卜和菜心致病力较弱。

关键词: 萝卜病毒病, 油菜花叶病毒, 分子特征, 侵染性克隆, 致病性

Abstract:

【Objective】Viral disease is one of the major factors threatening the production safety and radish farming industry. The objective of this study is to identify the viruses that infect radish in Guangdong Province, and to analyze the molecular characteristics and pathogenicity of youcai mosaic virus (YoMV), so as to provide theoretical basis for the prevention of viral disease in radish. 【Method】Three symptomatic and one asymptomatic samples were collected from a radish-planting base in Guangdong and were subjected to total RNA extraction. Small RNA sequencing and assembly (sRSA) was used to identify the virus species, followed by RT-PCR verification with specific primer pairs designed according to the sRSA results. To construct the infectious cDNA clones of YoMV-GD isolate, two primer pairs were designed, and the PCR products were introduced into pCB301 vector by seamless cloning and assembly. Then the full-length genomic sequences of YoMV-GD and other YoMV isolates were used to construct phylogenetic tree by MEGA7 software. Agrobacterium containing pCB301-YoMV-GD was infiltrated into Nicotiana benthamiana and Nicotiana tabacum to verify the infectivity of YoMV-GD infectious cDNA clones. Subsequently, the pathogenicity of YoMV-GD was tested in radish and Chinese flowering cabbage by agroinfiltration. 【Result】sRSA result showed that the viruses that infecting radish in Guangdong include Raphanus sativus cryptic virus 3 (RsCV3), Raphanus sativus chrysovirus 1 (RasCV1), turnip mosaic virus (TuMV), and YoMV. Sanger sequencing reveals that YoMV-GD isolate contains 6 304 nt and encodes four proteins, replication-associated protein, replicase, movement protein, and coat protein. YoMV-GD shares the highest similarity (99.18%) with YoMV British isolate (GenBank accession number: AY318866). Phylogenetic analysis shows that YoMV-GD has a closer relationship with isolates from British, Germany, Shanghai, and Chongqing. In addition, the YoMV-GD infectious cDNA clones could successfully infect N. benthamiana and N. tabacum, causing crinkle, mosaic, and necrosis symptoms in the upper leaves. Moreover, though YoMV-GD isolate could systemically infect radish and Chinese flowering cabbage, the symptoms were very weak. 【Conclusion】Viruses infecting radish in Guangdong include RsCV3, RasCV1, TuMV, and YoMV, and mixed infection is the main type. There was no clear regional difference among YoMV isolates. YoMV-GD is highly pathogenic in tobacco, but weak in radish and Chinese flowering cabbage.

Key words: viral disease in radish, youcai mosaic virus (YoMV), molecular characteristics, infectious clone, pathogenicity

李正刚,汤亚飞,佘小漫,于琳,蓝国兵,何自福. 侵染萝卜的油菜花叶病毒广东分离物分子特征及其致病性分析[J]. 中国农业科学, 2022, 55(14): 2752-2761.

LI ZhengGang,TANG YaFei,SHE XiaoMan,YU Lin,LAN GuoBing,HE ZiFu. Molecular Characteristics and Pathogenicity Analysis of Youcai Mosaic Virus Guangdong Isolate Infecting Radish[J]. Scientia Agricultura Sinica, 2022, 55(14): 2752-2761.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2022.14.006

https://www.chinaagrisci.com/CN/Y2022/V55/I14/2752

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引物名称 Primer name	引物序列 Primer sequence	对应位置 Localization (nt)	产物长度 Product length (bp)	退火温度 Annealing temperature (℃)
RsCV3-F	ACAGGATTCATCTCGGATCTT	140-160	1042	55
RsCV3-R	ACTAGTGAATCGTCTCCTAATGT	1182-1160	1042	55
RasCV1-F	AATGTGATTGTCACACATGAACTAG	498-522	1030	55
RasCV1-R	CTAATAGCCCCATCCTTCAGG	1528-1508	1030	55
YoMV-F	AATATCAGAGAGCGGCATTCTAT	550-572	1104	55
YoMV-R	CTGTCGGTAAAAGTGCAATATAACT	1654-1630	1104	55
TuMV-F	GTCGCCGTTCTGTTGGTCGTTTTGA	1509-1533	1371	55
TuMV-R	TGCCTGCGATAACTGCTTGTGTACG	2879-2855	1371	55
301-YoMV-F1	AAGTTCATTTCATTTGGAGAGGGTT TTATTTTTATTGCAACAACAACAA	1-27	3405	66
301-YoMV-R1	TGAACACTGTGTCGATCTGTAATT GCTATTGGGTCCCCGA	3405-3366	3405	66
301-YoMV-F2	AACAGCGACTCCCTTGGAGATCAT ATCAAGAGCGTCACCTCATG	3194-3237	3111	70
301-YoMV-R2	GGTGGAGATGCCATGCCGACCCTT GGGCCCCTACCCGGGGTTA	6304-6284	3111	70

基因登录号 GenBank number	长度 Length (bp)	覆盖度 Coverage (%)	拼接序列条数 Contig	测序深度 Depth	平均测序深度 Depth (Norm)	同源性 Identity (%)	最高同源性 Iden. Max (%)	最低同源性 Iden. Min (%)	科 Family	属 Genus	种 Species
FJ461349	1609	1544 (96.0)	1	82.0	6.8	99.42	99.42	99.42	Partitiviridae	maybe Alphapartitivirus	RsCV3 segment 1
FJ461350	1581	1404 (88.8)	12	19.9	1.7	99.50	100.00	95.56	Partitiviridae	maybe Alphapartitivirus	RsCV3 segment 2
JQ045335	3638	3411 (93.8)	12	28.7	2.4	98.83	100.00	97.62	Chrysoviridae	Alphachrysovirus	RasCV1 segment 1
JQ045336	3517	3419 (97.2)	6	21.5	1.8	98.86	99.38	97.73	Chrysoviridae	Alphachrysovirus	RasCV1 segment 2
JQ045337	3299	3218 (97.5)	9	31.6	2.6	99.25	100.00	97.91	Chrysoviridae	Alphachrysovirus	RasCV1 segment 3
EU734433	9833	9833 (100)	127	2343.0	194.7	95.33	100.00	89.17	Potyviridae	Potyvirus	TuMV isolate TANX2
AY318866	6304	6302 (100)	1	980.2	81.4	99.21	99.21	99.21	Virgaviridae	Tobamovirus	YoMV