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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 (ZYMV) is a common virus that damages Guangdong wax gourd. The optimal extraction method of total RNA from leaves of wax gourd was explored. Sixty-six virus-infected samples collected from nine winter melon producing areas in Guangdong Province were detected by RT-PCR based on the coat protein gene (cp) sequences of ZYMV virus. Of the incidence of the virus in the field; the amplification product was cloned and sequenced, the phylogenetic tree was constructed using MegAlign software to analyze the genetic variation and phylogeny of cp gene. The results showed that 24 samples were positive and the incidence rate in the field was 31.59%. The cp gene of all the tested virus isolates was 840 bp in length and encoded a polypeptide of 279 amino acids. The nucleotide sequences of cp gene of 16 isolates were sequenced The deduced amino acid sequences of all Guangdong isolates were conserved with the deduced amino acid sequences of 98.1% -100% and 96.1% -100%, respectively. All GD isolates contained 5 human leukocyte antigens except GD121-9. The human leukocyte antigen and one N-glycosylation site all contained the conserved potyv_CP domain and the domain DAG triplex box “Asp-Ala-Gly ” which related to the virus aphid. Phylogenetic analysis showed that the ZYMV virus partition Seven genotypes, Guangdong isolates belonged to the same genotype Ⅰ and consisted of three strains. Sixteen isolates from Guangdong were divided into three groups with no obvious regional selectivity. In general, Guangdong ZYMV-cp is more conservative with smaller molecular variance. The infection status of ZYMV virus on wax gourd in Guangdong Province and the variation characteristics of the coat protein gene were clarified to provide the basis for the study of its pathogenicity and antiviral gene engineering.