广东地区冬瓜感染的小西葫芦黄花叶病毒检测及其外壳蛋白基因多样性分析

Detection of Zucchini yellow mosaic virus Infecting Wax Gourd in Guangdong Province and Genetic Variation of Its Coat Protein Gene

小西葫芦黄花叶病毒（Zucchini yellow mosaic virus,ZYMV）是危害广东省冬瓜的常见病毒。摸索了冬瓜叶片总RNA最佳提取方法,通过基于ZYMV病毒外壳蛋白基因（coat protein gene,cp）序列的RT-PCR扩增,检测采集自广东省9个冬瓜产区的76份病毒侵染样品的病毒田间发病率;将扩增产物克隆测序,利用MegAlign软件构建系统发育进化树分析cp基因遗传变异与系统进化。结果表明,24份样品检测为阳性,田间发病率为31.59%;所有参试病毒分离物cp基因全长840 bp,编码279个氨基酸的多肽;测序的16个分离物cp基因的核苷酸序列与推导蛋白的氨基酸序列相似性分别为98.1%~100%与96.1%~100%;所有广东分离物的推导氨基酸序列基序保守,除GD121-9增加2个人类白细胞抗原外,其他均包含5个人类白细胞抗原及1个N糖基化位点,均包含保守potyv_CP功能域及与病毒蚜传相关的结构域DAG三联盒＂Asp-Ala-Gly＂;系统进化分析表明,ZYMV病毒划分为7个基因型,广东分离物均属于同一基因型Ⅰ,包含3个株系;广东地区的16个分离物分为3组,无明显地域选择性。总体上广东ZYMV-cp比较保守,分子变异较小。明确了广东省冬瓜上ZYMV病毒的侵染情况及外壳蛋白基因的变异特点,为对其致病性和抗病毒基因工程等研究提供依据。

Zucchini yellow mosaic virus was a common virus infecting wax gourd in Guangdong Province. Total RNA extraction methods of wax gourd leaves were compared,and then 76 wax gourd samples from 9 main production regions in Guangdong Province were collected and detected based on RT-PCR amplification of coat protein gene（cp）of ZYMV. The amplification products were sequenced and genetic variation analysis were carried out. The results showed that 24 samples were determined as positive with incidence ratio of 31.59%,sequence analysis revealed that full-length of ZYMV-cp of 16 isolates was 840 bp encoding a peptide with 279 amino acids. Further alignment analysis indicated that similarities of ZYMV-cp nucleotide acid and amino acid sequences ranged from 98.1% to 100% and from 96.1% to 100% respectively. The motifs of deduced amino acids sequences were conserved,all isolates contained 5 HLA sites（Human Leukocyte Antigen）and 1 N-glycosylation site expect GD121-9 with 2 additive HLA motifs. The CP protein of all isolates included conserved domain potyv_CP and DAG triple box（Asp-Ala-Gly）crucial for potyvirus transmission by aphid. Phylogenetic analysis suggested that all isolates were clustered into 7 genotypes,Guangdong isolates belonged to genotype I and contained 3 strains;All Guangdong isolates were further clustered into 3 groups with no obvious geographical differentiation. Generally ZYMV-cp in Guangdong isolates were conserved and had little molecular variation. This study determined the infection situation and genetic variation of coat protein gene of ZYMV isolates in Guangdong Province,and laid foundation for further pathogenicity research and antivirus genetic engineering.