江苏黄瓜绿斑驳花叶病毒的鉴定

Identification of cucumber green mottle mosaic virus in Jiangsu

为确定从江苏省洪泽县大棚西瓜、仪征市露地瓠瓜、东台市露地南瓜上采集到3个表现为褪绿、斑驳花叶症状的病样是否为黄瓜绿斑驳花叶病毒(CGMMV)侵染,采用电子镜观察、RT-PCR和序列测定方法对其进行鉴定。电子显微镜下可见300 nm×18 nm大小的直杆状病毒粒子。根据已报道的CGMMV外壳蛋白质(cp)基因序列合成特异性引物,对所提取病样的总RNA进行RT-PCR扩增,3个分离物的RNA模板中均可扩增到大小为500 bp左右的DNA片段,测序结果表明3个分离物的片段均含有486个核苷酸,包含完整的cp基因,编码161个氨基酸。3个分离物与已报道的7个CGMMV分离物核苷酸及其所推导的氨基酸序列同源性分别为91.8%～99.8%和98.1%～100.0%,与同属的其他3个病毒(Kyuri绿斑驳花叶病毒、黄瓜果实斑驳花叶病毒和小西葫芦绿斑驳花叶病毒)cp基因核苷酸及其所推导的氨基酸序列同源性分别为40.7%～54.7%和44.4%～60.2%,表明所采集的3个病样由CGMMV侵染引起。

This study was performed to identify three isolates with chlorisis and mottle mosaic symptom collected from greenhouse watermelon in Hongze of Jiangsu, openground gourd in Yizheng of Jiangsu and openground pumpkin in Dongtai of Jiangsu. Straight rod virus particles with size of 300 nm×l8 nm were shown under the electron micrograph in the crude extract of above isolates. Total RNA were amplified by RT-PCR with the specific primers designed on the basis of the reported coat protein （cp） gene of cucumber green mottle mosaic virus（ CGMMV）. An 500-bp gene fragment was amplified from total RNA of all three isolates, and subsequent sequence analysis indicated that the 486 hp fragment contained complete cp gene which encoded 161 amino acids. The nucleotides and deduced amino acid sequences of the cp genes from these isolates shared homologies from 91.8% to 99. 8% and from 98. 1% to 100. 0% with 7 reported CGMMV, respectively, and shared homologies from 40. 7% to 54. 7% and from 44.4% to 60. 2% with other 3 Tobamovirus, Kyuri green motfie mosaic virus （KGMMV）, cucumber fruit mottle mosaic virus （CFMMV） and zucchi green mottle mosaic virus （ZGM-MV）, respectively. All above indicated that three collected samples were infected by CGMMV.