银川番茄斑萎病毒的分子鉴定

doi:10.11983/CBB21113

植物学报 ›› 2021, Vol. 56 ›› Issue (6): 715-721.DOI: 10.11983/CBB21113

银川番茄斑萎病毒的分子鉴定

王晓敏¹^,²^,³^,⁴^,^*(), 李洪磊¹, 王林¹, 周鹏泽¹, 白圣懿¹, 李国花¹, 郑福顺¹, 陶小荣⁵, 程国新¹^,²^,³^,⁴, 高艳明¹^,²^,³^,⁴, 李建设¹^,²^,³^,⁴

¹宁夏大学农学院, 银川 750021
²宁夏现代设施园艺工程技术研究中心, 银川 750021
³宁夏优势特色作物现代分子育种重点实验室, 银川 750021
⁴宁夏设施园艺(宁夏大学)技术创新中心, 银川 750021
⁵南京农业大学植物保护学院, 南京 210095

收稿日期:2021-07-09 接受日期:2021-11-20 出版日期:2021-11-01 发布日期:2021-11-17
通讯作者: 王晓敏
作者简介:* E-mail: wangxiaomin_1981@163.com
基金资助:
宁夏回族自治区农业特色优势产业育种专项(NXNYYZ20200101);宁夏回族自治区重点研发计划(2021BBF02002);宁夏回族自治区重点研发计划(重大)重点项目(2019BBF02022)

Molecular Identification of Tomato Spotted Wilt Virus on Tomato in Yinchuan

Xiaomin Wang¹^,²^,³^,⁴^,^*(), Honglei Li¹, Lin Wang¹, Pengze Zhou¹, Shengyi Bai¹, Guohua Li¹, Fushun Zheng¹, Xiaorong Tao⁵, Guoxin Cheng¹^,²^,³^,⁴, Yanming Gao¹^,²^,³^,⁴, Jianshe Li¹^,²^,³^,⁴

¹School of Agriculture, Ningxia University, Yinchuan 750021, China
²Ningxia Modern Facility Horticulture Engineering Technology Research Center, Yinchuan 750021, China
³Key Laboratory of Modern Molecular Breeding for Dominant and Special Crops in Ningxia, Yinchuan 750021, China
⁴Ningxia Facility Horticulture (Ningxia University) Technology Innovation Center, Yinchuan 750021, China
⁵College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China

Received:2021-07-09 Accepted:2021-11-20 Online:2021-11-01 Published:2021-11-17
Contact: Xiaomin Wang

摘要/Abstract

摘要： 为明确银川番茄(Lycopersicon esculentum)是否遭受了番茄斑萎病毒(TSWV)的危害, 采用国家标准TSWV RT- PCR检测技术对银川番茄上采集的14份疑似感染TSWV病叶样本进行分子鉴定, 对克隆得到的核衣壳蛋白基因N (Nucleocapsid)序列进行多序列比对和系统进化树分析, 随后对PCR阳性样本进行蛋白检测。结果表明, 14份病叶样本中有8份扩增出长度为394 bp的TSWV N基因序列, 且8条序列完全一致; 获得的银川番茄TSWV分离物与云南番茄、中国莴苣(Lactuca sativa)、中国鸢尾(Iris tectorum)和重庆辣椒(Capsicum annuum) TSWV分离物相对近缘, 与山东、黑龙江和北京等地及国外TSWV分离物相对远缘; 利用TSWV的抗体通过Western blot对8个PCR阳性样本进一步检测, 结果也证实8个阳性样本中存在TSWV感染。该研究首次通过分子鉴定及蛋白检测证明银川番茄上存在TSWV感染, 需要加快抗TSWV番茄品种的选育工作。

关键词: 银川, 番茄斑萎病毒, RT-PCR, Western blot

Abstract: In order to determine whether Yinchuan tomato (Lycopersicon esculentum) was damaged by tomato spotted wilt virus (TSWV), 14 disease leaf samples of suspected infected TSWV collected from Yinchuan tomato were identified by the national standard TSWV RT-PCR technique. The cloned N gene sequences were analyzed by multi sequence alignment and phylogenetic tree analysis. Eight of the 14 disease leaf samples amplified the TSWV N gene sequence with a length of 394 bp, and the 8 sequences were completely consistent. The TSWV sequence was relatively close to those from Yunnan tomato, Chinese lettuce (Lactuca sativa), Chinese Iris (Iris tectorum) and Chongqing pepper (Capsicum annuum), while relatively distant to those from Shandong, Heilongjiang, Beijing and the abroad. Detection of TSWV in the 8 PCR positive samples by use of Western bolt showed that TSWV infection existed in these samples. This study proved that there was TSWV infection in Yinchuan tomato. Therefore, it is necessary to speed up the breeding of TSWV resistant tomato varieties.

Key words: Yinchuan, tomato spotted wilt virus, RT-PCR, Western blot

王晓敏, 李洪磊, 王林, 周鹏泽, 白圣懿, 李国花, 郑福顺, 陶小荣, 程国新, 高艳明, 李建设. 银川番茄斑萎病毒的分子鉴定. 植物学报, 2021, 56(6): 715-721.

Xiaomin Wang, Honglei Li, Lin Wang, Pengze Zhou, Shengyi Bai, Guohua Li, Fushun Zheng, Xiaorong Tao, Guoxin Cheng, Yanming Gao, Jianshe Li. Molecular Identification of Tomato Spotted Wilt Virus on Tomato in Yinchuan. Chinese Bulletin of Botany, 2021, 56(6): 715-721.

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https://www.chinbullbotany.com/CN/Y2021/V56/I6/715

图/表 3

图1 番茄田间采集病样症状及TSWV的RT-PCR检测 (A) 叶片出现黑斑; (B) 果实伴有黄化和畸形; (C) 植株萎蔫; (D) 病样TSWV的RT-PCR检测(M: DL2000 marker; CK+: 阳性对照; CK-: 阴性对照; 1-14: 番茄病叶样本)。Bars=10 cm

Figure 1 RT-PCR detection of TSWV and disease symptoms in tomato field (A) Black spots appeared on the leaves; (B) The fruits sho-wing yellowing and deformity; (C) Wilted plant; (D) Detection of TSWV in disease samples by RT-PCR (M: DL2000 mar-ker; CK+: Positive control; CK-: Negative control; 1-14: To-mato diseased leaf samples). Bars=10 cm

图2 基于N基因序列一致性的片段多序列比对(A)和系统进化树分析(B) *: 本研究N基因序列。Bar=0.005

Figure 2 Multiple sequence alignment (A) and phylogenetic tree analysis (B) based on sequence consistency of N gene *: N gene sequence of this study. Bar=0.005

图3 番茄斑萎病毒的Western blot检测 M: 分子量蛋白marker; CK-: 阴性对照; 1、3、4、8、9、10、11和14: 检测样本

Figure 3 Western blot analysis of tomato spotted wilt virus M: Protein size marker; CK-: Negative control; 1, 3, 4, 8, 9, 10, 11 and 14: Test samples

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银川番茄斑萎病毒的分子鉴定

Molecular Identification of Tomato Spotted Wilt Virus on Tomato in Yinchuan

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