浙江菜豆CPMMV变异株的鉴定及其TGB蛋白定位

doi:10.16420/j.issn.0513-353x.2023-0801

摘要/Abstract

摘要：

2022年10—11月，在浙江省杭州市钱塘区观察到疑似病毒侵染的菜豆植株，病毒组测序和PCR技术检测出豇豆轻斑驳病毒（cowpea mild mottle virus，CPMMV）、黄瓜花叶病毒（cucumber mosaic virus，CMV）和紫云英矮缩病毒（milk vetch dwarf virus，MDV），并将CPMMV命名为CPMMV-ZJ（GenBank：OR667247）。利用病毒鉴定、全基因组序列扩增、系统发育分析及亚细胞定位等方法，明确了CPMMV-ZJ的全基因组序列特征、病毒演化及其病毒三联蛋白（triple gene block protein，TGB）的亚细胞定位。结果表明，CPMMV-ZJ的基因组全长为8 226 bp，包含6个开放阅读框。3′和5′区域分别有1个147 bp和75 bp的非编码区（untranslated regions，UTR）。序列分析结果表明，CPMMV浙江分离物与CPMMV安徽分离物的全基因组序列一致性最高（98.03%），并与国内分离物共同聚类在一个小分支上，但与国外分离物（美国、印度、巴西等地）序列一致性较低（63.71% ~ 82.49%），提示病毒变异与地理因素密切相关。对浙江分离物与江苏分离物TGB蛋白的亚细胞定位比较分析发现，二者无明显差异，但TGB2浙江分离物在叶片中的表达导致明显的叶绿体核周聚集现象，推测与植物防御反应有关。

关键词: 菜豆, 豇豆轻斑驳病毒, 序列分析, 亚细胞定位, 叶绿体核周聚集

Abstract:

During the period from October to November 2022，an outbreak of suspected virus-infected common bean occurred in the Qiantang District of Hangzhou City，Zhejiang Province. Subsequent viral genome sequencing and PCR confirmed the presence of cowpea mild mottle virus（CPMMV），cucumber mosaic virus（CMV），milk vetch dwarf virus（MDV）. CPMMV was designated as CPMMV-ZJ（GenBank：OR667247）. By virus identification，genome sequence amplification，phylogenetic analysis and subcellular localization analysis，the sequence structure of CPMMV-ZJ and the localization of the triple gene block proteins（TGB）were elucidated. The genome of CPMMV-ZJ encompassed a total length of 8 226 bp，consisting of six open reading frames. Additionally，both the 3′ and 5′ regions contained non-coding regions（UTRs）of 147 and 75 bp，respectively. Phylogenetic analysis of CPMMV-ZJ revealed the highest sequence identity（98.03%）with the CPMMV Anhui isolate，whereas lower identities（63.71%-82.49%）were observed when comparing to foreign isolates from the United States，India，and Brazil，indicating virus evolution is related to geographical factors. Subcellular localization analysis demonstrated similar localization of TGBs between the Zhejiang and Jiangsu isolates. Notably，overexpression TGB2 of the Zhejiang isolate leads to a clustering of chloroplasts around the nuclei，suggesting its potential involvement in plant defense responses.

Key words: common bean, cowpea mild mottle virus, sequence analysis, subcellular localization, clustering of chloroplasts around the nuclei

张子鑫, 陈淑婷, 周伟欣, 张意涵, 鲁倩, 李中义, 吴昕扬, 徐沛. 浙江菜豆CPMMV变异株的鉴定及其TGB蛋白定位[J]. 园艺学报, 2025, 52(1): 123-135.

ZHANG Zixin, CHEN Shuting, ZHOU Weixin, ZHANG Yihan, LU Qian, LI Zhongyi, WU Xinyang, XU Pei. Identification of Cowpea Mild Mottle Virus Variants in Zhejiang and Localization of TGB Proteins[J]. Acta Horticulturae Sinica, 2025, 52(1): 123-135.

图/表 9

表1 本试验中所用到的引物

Table 1 Primers sequences used in this study

用途 Usage	引物 Primer	引物序列（5′-3′） Primers sequence
5′端快速cDNA扩增 5′RACE	5AP	GGTCTCAAGGGCTCTAAACATTT
	GSP1	CAACTCATGACCAGCTCAGC
	GSP2	TTCCCTTGGCAGAACGAGAC
序列扩增 Sequence amplification	CPMMV-F1	GTTTACCCACCGGAGTTGCT
	CPMMV-R2	AATCACTTTGGCAGGGATCC
	CPMMV-F2	TCAAAGCTAGAATGTCTAGG
序列扩增；病毒检测 Sequence amplification；Virus detection	CPMMV-R2	CTGTCAATACAGTTTTGGAG
序列扩增；病毒检测 Sequence amplification；Virus detection	CPMMV-F3	AGATGAAGGAATTAGAGAGG
序列扩增 Sequence amplification	CPMMV-R3	AGAACCTCAGCTACGTGCTC
3′端快速cDNA扩增 3′RACE	3AP	GTTTTCCCAGTCACGACAC
	GSP3	GCCCTTCCAGACATTGATGC
	GSP4	TCAAATAACATGGCCACAGCTG
亚细胞定位载体构建 Subcellular localization vector construction	TGB1-F	ATGAATGAACTGATCAGTAA
	TGB1-R	CTCAGAGTTTGGATAGGTTG
	TGB2-F	ATGCCACTGACTCCACCA
	TGB2-R	GTGAACCCTATTGCAGA
	TGB3-F	ATGTCTGCAATAGGGTTCAC
	TGB3-R	CAACCTACAACTTAGGCTA
病毒检测 Virus detection	MDV-F	ACTCAAGGAGAGGCAAGAGC
	MDV-R	ATGTCGGCTGTCTTTCCACC
	CMV-F	AAGTGGTTTGCAGCGTTGAC
	CMV-R	TGTTGCAAATCACGCACTCG

图1 菜豆病毒病田间症状

Fig. 1 Filed symptoms of common bean infected with virus diseases

图2 菜豆田间样品中CPMMV、CMV、MDV的RT-PCR检测 M：DL2 000 DNA marker；1 ~ 10：随机挑选的10份田间感染病毒的菜豆叶片样品

Fig. 2 RT-PCR detection of CPMMV，CMV and MDV from samples randomly selected in field of common bean M：DL2 000 DNA marker；1-10：Ten randomly selected common bean leaf samples from virus-infected field plants

图3 菜豆叶片机械接种CPMMV-ZJ（7 d）后的症状

Fig. 3 Symptoms of common bean leaves mechanically inoculated with CPMMV-ZJ at 7 days post inoculation

图4 CPMMV-ZJ机械接种健康菜豆叶片后的RT-PCR检测 M：DL2 000 DNA marker；1 ~ 10：接种CPMMV的菜豆叶片样品；11：阳性对照；12：阴性对照

Fig. 4 RT-PCR detection of CPMMV in common bean leaves mechanically inoculated with CPMMV-ZJ M：DL2 000 DNA marker；1-10：Common bean leaf samples inoculated with CPMMV；11：Positive control；12：Negative control

图5 CPMMV-ZJ基因组分段扩增策略

Fig. 5 Genome segmentation amplification strategies for CPMMV-ZJ

图6 菜豆CPMMV浙江分离物（CPMMV-ZJ）基因组结构

Fig. 6 Genomic structure of the CPMMV Zhejiang isolate（CPMMV-ZJ）from common bean

图7 菜豆CPMMV浙江分离物（CPMMV-ZJ）系统发育分析图分支上的数字表示自举值

Fig. 7 Phylogenetic analysis of CPMMV-ZJ The numbers on the branches of the tree represent bootstraps

图8 CPMMV浙江分离物（CPMMV-ZJ）与江苏分离物（CPMMV-JS）的TGB蛋白亚细胞定位（A）及TGB2-ZJ引起的叶绿体核周聚集现象（B）白色箭头指示叶绿体在核周聚集

Fig. 8 Subcellular localization analysis of TGBs of CPMMV Zhejiang（CPMMV-ZJ）and Jiangsu（CPMMV-JS）isolate（A）and the clustering of chloroplasts around the nuclei caused by TGB2-ZJ（B） White arrows represents perinuclear aggregation of chloroplasts

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