黄瓜绿斑驳花叶病毒基因组全序列分析及病害的田间调查

为明确近年来在浙江省葫芦科作物上发生的黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus,CGMMV)基因组特征及其发生分布情况,从浙江省及上海地区的甜瓜、西瓜和瓠瓜上采集疑似样品进行RT-PCR鉴定,通过分段扩增测序的方法拼接获得基因组全序列并进行系统进化分析,利用特异性引物扩增获得CGMMV外壳蛋白(coat protein,CP)基因序列,制备CGMMV CP抗血清进行Western-blot和Dot-ELISA检测。结果显示,来自甜瓜、西瓜和瓠瓜的3个CGMMV分离物基因组全序列均具有烟草花叶病毒属典型基因组结构特征,全部由6 423 nt构成;3个全序列间的核苷酸同源性高达99.11%~99.67%,编码的CP氨基酸同源性为100%。系统进化分析发现,CGMMV不同分离物形成2个进化相关群体,3个浙江的CGMMV分离物均位于第I组内,与已报道的中国CGMMV分离物和韩国CGMMV分离物亲缘性较高。Western-blot检测表明CGMMV CP抗血清可以与感病植株中的病毒发生特异性反应,可用于CGMMV鉴定;Dot-ELISA检测发现CGMMV在浙江省和上海市的葫芦科作物上普遍存在。

The complete genomic sequence analysis of three isolates of Cucumber green mottle mosaic virus in Zhejiang and the application of prepared antiserum

To understand the molecular character of Cucumber green mottle mosaic virus(CGMMV) occurred in Zhejiang Province, three CGMMV infected samples showing mottle, mosaic and distort symptoms on leaf were collected from melon, watermelon and bottle gourd for analysis. The complete genomic RNA sequences of the three CGMMV isolates were determined by assembling two fragments amplified from these samples by RT-PCR which overlapped the whole CGMMV genome. For surveying the occurrence and distribution of CGMMV in Zhejiang and Shanghai, the coat protein of CGMMV expressed with E. coli was purified and used for preparation of antiserum against the virus. Results revealed that each of three CGMMV isolates was consisted of 6423 nucleotides with typical character of the genome of genus Tobamovirus. Multiple sequence alignments of genomes of these isolates showed that identities of the nucleotide sequences were 99.11%-99.67%, while the coat protein amino acid sequences were identical. Phylogenetic tree generated from CGMMV complete nucleotide sequences available suggested that CGMMV could be separated into two subgroups. The three Zhejiang isolates determined in this work belonged to subgroup I, and the genetic relationship of these Zhejiang isolates was close to that of the Korean CGMMV isolates. Western-blot result suggested that the antiserum could specifically react with the CGMMV infected plants. By using the specific antiserum to investigate the CGMMV on the cucurbit plants in fields, it was found that CGMMV infection on cucurbit crops was common in fields in Zhejiang Province and Shanghai City.