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

MA Xin-yan; GU Qin-sheng; LIU Li-feng; LI Ning; YANG Min-he; PENG Bin; LI Li

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June 2005

MA Xin-yan, GU Qin-sheng, LIU Li-feng, LI Ning, YANG Min-he, PENG Bin and LI Li. Cloning and Construction of Plant Expression of the Coat Protein Gene of a Melon Isolate of Cucumber Mosaic Virus[J]. Virologica Sinica, 2005, 20(3): 307-310.

Citation: MA Xin-yan, GU Qin-sheng, LIU Li-feng, LI Ning, YANG Min-he, PENG Bin, LI Li. Cloning and Construction of Plant Expression of the Coat Protein Gene of a Melon Isolate of Cucumber Mosaic Virus .VIROLOGICA SINICA, 2005, 20(3) : 307-310.

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

马新艳 ^1,2 ,
古勤生 ^1* ,
刘丽锋 ¹ ,
李宁 ¹ ,
杨民和 ³ ,
彭斌 ¹ ,
李莉 ¹

1.
1.中国农业科学院郑州果树研究所生物技术中心河南郑州 2.江西农业大学农学院江西南昌 3.福建福州福建师范大学生物工程学院

摘要

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。
- CMV
- , 中国分离物
- , 外壳蛋白基因
- , 序列分析
- , 植物表达载体

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

MA Xin-yan ^1,2 ,
GU Qin-sheng ^1* ,
LIU Li-feng ¹ ,
LI Ning ¹ ,
YANG Min-he ³ ,
PENG Bin ¹ ,
LI Li ¹

Abstract

The coat protein gene of Cucumber mosaic virus (CMV) from melon isolate (CH99/ 90) was amplified by RT2PCR from the total RNA of infected zucchini leaves and cloned into pUCm2 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 wit h 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 tume faciens LBA4404. Transformation of the gene into watermelon is undertaking.
- CMV
- , Chinese isolate
- , Coat protein gene
- , Sequence analysis
- , Plant expression vector

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

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

Abstract: The coat protein gene of Cucumber mosaic virus (CMV) from melon isolate (CH99/ 90) was amplified by RT2PCR from the total RNA of infected zucchini leaves and cloned into pUCm2 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 wit h 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 tume faciens LBA4404. Transformation of the gene into watermelon is undertaking.