CMV甜瓜分离物外壳蛋白基因的克隆及植物表达载体的构建

Cloning and Construction of Plant Expression of the Coat Protein Gene of a Melon Isolate of Cucumber Mosaic Virus

从河南省临颖县采集的病毒感染的甜瓜样本经ELISA检测和接种分离获得黄瓜花叶病毒(Cucunbermosaicvirus,CMV)分离物。把该分离物接种西葫芦,从发病的叶片中提取总RNA,并以此为模板经RTPCR扩增获得CMV的外壳蛋白(cp)基因,将其克隆到pUCmT质粒上。经序列测定和分析,结果表明该cp基因由657个核苷酸组成,编码218个氨基酸。其核苷酸序列与黄瓜花叶病毒亚组I的分离物有较高的同源性,达92.2%～93.9%,与亚组II的同源性仅为76.8%～77.8%,与我国报道的CMV分离物的cp基因序列比较,除香蕉株系XB外核苷酸序列的同源性达91.8%～93.4%。根据这些分析,该CMV分离物属于亚组I。将cp基因通过中间载体pJIT163定向克隆到植物表达载体pBINPLUS中(重组双元载体质粒命名为pBCP),并经冻融法导入农杆菌中,经PCR及酶切鉴定,证实质粒已被导入。利用该植物表达载体对西瓜的遗传转化工作目前正在进行中。

The coat protein gene of Cucumber mosaic virus(CMV)from melon isolate (CH99/90) was amplified by RT-PCR from the total RNA of infected zucchini leaves and cloned into pUCm-T vector. The gene consisted of 657 nucleotides encoding 218 putative amino acids. Nucleotide sequence alignments showed that the CP gene shared 92.2%~93.9% homology with that of CMV subgroup I strains, whereas only 76.8%~77.8% homology with that of CMV subgroup II strains.The nucleotide sequence shared 91.8%~93.4% homology with CMV isolate previously reported from China except for XB isolate. This melon isolate was classified into subgroup I based on these data. The gene was constructed into plant expression vector by using intermediate vector pJIT163 (recombinant plasmid designated as pBCP). The recombinant plasmid was transferred into competent cells of Agrobacterium tumefaciens LBA4404. Transformation of the gene into watermelon is undertaking.