侵染广东连州葫芦的黄瓜绿斑驳花叶病毒的分子特征 及致病性分析

doi:10.3864/j.issn.0578-1752.2020.05.008

摘要/Abstract

摘要：

【目的】黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus,CGMMV)是侵染瓜类作物的主要病毒之一,对瓜类产业造成巨大的危害。本研究旨在探明侵染广东省连州市葫芦的CGMMV分离物(CGMMV-GDLZ)分子特征及其在系统进化中的地位,并测定其对黄瓜、葫芦和西瓜的致病性,为CGMMV的防控提供理论依据。【方法】从广东省连州市葫芦种植基地采集2个疑似CGMMV侵染的病样以及1个无症状样品,提取总RNA,根据CGMMV参考序列（GenBank登录号：KX883801）设计引物进行RT-PCR检测,引物序列为F：CCACGAGTTGTTTCCTAATGCTG/R：TTTGCTAGGCGTGATCGGATTGT,退火温度53℃,扩增长度890 bp。将CGMMV全长序列分为前后两段,前半段1—3 511 nt,扩增引物序列为F：AAGTTCATTTCATTTGGAGAGGGTTTTAATTTTTATAA TTAAACAAA/R：AGTTCTGCATTAATTGCTATTTGGTAGGCACAGTGGTAG;后半段3 301—6 423 nt,扩增引物序列为F：GTGCGTGCTACCCCGACTCCAATAGGTTTGATTGCCCGTG/R：GGTGGAGATGCCATGCCGACCCTGGGCCCCTACCCGGGGAAAGG。将前后两段PCR产物通过同源重组的方法克隆到pCB301双元载体上,测序得到CGMMV-GDLZ分离物全长序列。利用CGMMV-GDLZ分离物全长序列在NCBI中进行Blast分析,然后通过MEGA7软件对CGMMV-GDLZ以及其他已经报道的CGMMV分离物进行系统进化树分析。将构建好的pCB301-CGMMV侵染性克隆注射接种本生烟验证其侵染性,然后再注射接种黄瓜、葫芦和西瓜的子叶,测定CGMMV-GDLZ分离物的致病性。【结果】RT-PCR结果证实,广东省连州市葫芦病样感染了CGMMV。CGMMV-GDLZ分离物全长序列为6 423 nt,编码4个蛋白,分别为129K复制酶（61—3 495 nt）、186K复制相关蛋白（61—5 007 nt）、运动蛋白MP（4 994—5 788 nt）和外壳蛋白CP（5 763—6 248 nt）。CGMMV-GDLZ核苷酸序列与CGMMV-eWT分离物（GenBank登录号：KY753928）同源性最高,为99.97%。系统进化树分析结果显示,CGMMV-GDLZ分离物与日本、韩国等东亚CGMMV分离物同属Group 1,在遗传距离上与山东、浙江和河南的CGMMV分离物最接近。pCB301-CGMMV侵染性克隆可以系统侵染本生烟,造成本生烟上部叶片出现皱缩、斑驳、凸起等症状,RT-PCR和Western blot进一步确认了侵染性克隆的侵染性。注射接种CGMMV-GDLZ分离物后15 dpi,葫芦和西瓜即可产生斑驳、花叶、突起、生长迟缓等症状,24 dpi时症状更明显。而15 dpi时,CGMMV-GDLZ分离物在黄瓜上的症状不明显,与未接种对照植株几乎没有区别;将植株从控温接种室移入网室中,30 dpi时,黄瓜植株上部叶片开始出现斑驳和花叶,40 dpi时,症状已经非常明显。RT-PCR和Western blot检测进一步确认了上述结果。【结论】侵染广东省连州市葫芦的CGMMV-GDLZ分离物与山东、浙江和河南的CGMMV分离物很可能具有相同的传染源;CGMMV-GDLZ分离物可以侵染本生烟、黄瓜、葫芦和西瓜等作物,但对这些作物的致病性存在差异。

关键词: 葫芦, 黄瓜绿斑驳花叶病毒, 分子特征, 侵染性克隆, 致病性

Abstract:

【Objective】Cucumber green mottle mosaic virus (CGMMV) is one of the main viruses that infect melon crops, which causes great harm to the melon industry. The objective of this study is to identify the molecular characteristic and phylogenetic status of CGMMV-GDLZ isolate, analyze the pathogenicity of CGMMV-GDLZ isolate on cucumber, bottle gourd, and watermelon, and to provide a theoretical basis for the prevention and control of CGMMV. 【Method】Two symptomatic samples and one asymptomatic sample were collected from a farm located in Lianzhou City of Guangdong Province and were subjected to total RNA extraction. RT-PCR detection was performed using primer pair F: CCACGAGTTGTTTCCTAATGCTG/R: TTTGCTAGGCGTGATCGGATTGT, which was designed according to CGMMV reference genome (GenBank accession number KX883801). The annealing temperature is 53℃, and the product is 890 bp in length. To construct pCB301-CGMMV infectious cDNA clones, two primer pairs were designed to amplify the first half (1-3 511 nt) and the second half (3 301-6 423 nt) of the nucleotide sequence of CGMMV-GDLZ isolate. The primer pair used to amplify the first half is F: AAGTTCATTTCATTTGGA GAGGGTTTTAATTTTTATAATTAAACAAA/R: AGTTCTGCATTAATTGCTATTTGGTAGGCACAGTGGTAG, and the primer pair used to amplify the second half is F: GTGCGTGCTACCCCGACTCCAATAGGTTTGATTGCCCGTG/R: GGTGGAGATGC CATGCCGACCCTGGGCCCCTACCCGGGGAAAGG. Then the acquired products were cloned into pCB301 by homologous recombination, followed by Sanger DNA sequencing. Blast analysis was performed in NCBI using the full-length sequence of CGMMV-GDLZ isolate, then the phylogenetic tree was constructed by MEGA7 software using the sequences of CGMMV-GDLZ and other reported CGMMV isolates. Agrobacterium containing pCB301-CGMMV was infiltrated into Nicotiana benthamiana leaves to verify the infectivity of CGMMV-GDLZ isolate. Subsequently, pCB301-CGMMV was agroinfiltrated into the cotyledon of cucumber, bottle gourd, and watermelon to analyze the pathogenicity of CGMMV. 【Result】RT-PCR detection verified that the two symptomatic samples were infected by CGMMV. Sanger sequencing reveals that CGMMV-GDLZ isolate contains 6 423 nt and encodes four proteins, 129K replicase (61-3 495 nt), 186K replication-associated protein (61-5 007 nt), movement protein (4 994-5 788 nt), and coat protein (5 763-6 248 nt). CGMMV-GDLZ isolate has the highest nucleotide similarity (99.97%) with CGMMV-eWT isolate (GenBank accession number KY753928). Phylogenetic analysis showed that CGMMV-GDLZ and other CGMMV isolates from Japan and Korea were clustered into Group 1, and CGMMV-GDLZ was closest to isolates from Shandong, Zhejiang, and Henan. In addition, the pCB301-CGMMV infectious cDNA clones could successfully infect N. benthamiana, causing crinkle, mottle, and mosaic symptoms in the upper leaves. RT-PCR and Western blot detection further verified the infection of the infectious cDNA clones. Moreover, CGMMV-GDLZ isolate could also infect bottle gourd and watermelon, causing mottle, mosaic, and growth retardation at 15 days post-infiltration (dpi), and showed more severe symptoms at 24 dpi. However, the upper leaves of cucumber agro-infiltrated with pCB301-CGMMV did not show obvious symptoms compared with control plants. Further studies found the agro-infiltrated plants began to appear mottle and mosaic symptoms at 30 dpi after transferred from the greenhouse to artificial net-room without temperature control. The symptoms were more obvious at 40 dpi. RT-PCR and Western blot detection further verified the above results. 【Conclusion】CGMMV-GDLZ isolate from Lianzhou City of Guangdong Province may have the same infection source with CGMMV isolates from Shandong, Zhejiang, and Henan provinces. CGMMV-GDLZ isolate can infect N. benthamiana, cucumber, bottle gourd, and watermelon, but the pathogenicity varies in different crops.

Key words: bottle gourd, Cucumber green mottle mosaic virus (CGMMV), molecular characteristic, infectious cDNA clones, pathogenicity

李正刚,农媛,汤亚飞,佘小漫,于琳,蓝国兵,邓铭光,何自福. 侵染广东连州葫芦的黄瓜绿斑驳花叶病毒的分子特征及致病性分析[J]. 中国农业科学, 2020, 53(5): 955-964.

LI ZhengGang,NONG Yuan,TANG YaFei,SHE XiaoMan,YU Lin,LAN GuoBing,DENG MingGuang,HE ZiFu. Molecular Characteristic and Pathogenicity Analyses of Cucumber green mottle mosaic virus (CGMMV) Infecting Bottle Gourd in Lianzhou, Guangdong[J]. Scientia Agricultura Sinica, 2020, 53(5): 955-964.

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