来源于桃和苹果的苹果褪绿叶斑病毒的部分分子生物学特性和cp基因的原核表达  

Partial molecular characterization of Apple chlorotic leaf spot virus from peach and apple trees and prokaryotic expression for cp gene

作　　者：郑银英[1] 王国平[1] 洪霓[1] 宋艳苏[1] 游红[1] 

机构地区：[1]华中农业大学植物科学技术学院

出　　处：《植物病理学报》2007年第4期356-361,共6页Acta Phytopathologica Sinica

基　　金：国家自然科学基金资助项目(30370997);科技部"863"课题资助项目(2001AA241142)

摘　　要：从桃和苹果上分离得到苹果褪绿叶斑病毒ACLSV-HBP和ACLSV-C2个分离物,采用RT-PCR法进行扩增,所获扩增片段经序列测定,其全长分别为1768nt(ACLSV-HBP)和1751nt(ACLSV-C)。这2个分离物扩增片段全长的同源性为83%,mp基因片段核苷酸和推导编码氨基酸序列同源性分别为82.6%和87.1%;cp基因均由582nt组成,其核苷酸和推导编码氨基酸序列同源性分别为87.8%和95.9%。将2个分离物的cp基因与已报道ACLSV分离物进行序列同源性比较,结果显示ACLSV-HBP与SX/2的cp基因核苷酸序列及推导编码氨基酸序列同源性最高,分别为94.0%和96.4%。将ACLSV-HBP分离物的cp基因克隆到原核表达载体pGEX-KG,在大肠杆菌BL21(DE3)中诱导表达,SDS-PAGE分析表明,融合蛋白大小约为46kDa。Western-blot分析表明,该基因在大肠杆菌内得到高效表达,融合蛋白具有抗原性。Two isolates of Apple chlorotic leaf spot virus were obtained from apple （ACLSV-C） and peach （ACLSV-HBP） trees grown in China. Nucleic acid fragments of size in 1 768 nt and 1 751 nt from ACLSVHBP and ACLSV-C, including 3＇-end region of mp gene, 582 nt CP-coding region and 5＇-end region of 3＇- NCR, were cloned and sequenced, respectively. Identity of the two nucleic acid fragments was 83%. The identities of partial nucleotide and deduced amino acid sequences from ACLSV-HBP and ACLSV-C were 82.6% and 87.1% for mp genes, 87.8% and 95.9% for cp genes. The sequence data obtained in this study were compared with reported sequences of ACLSV isolates from different sources. The results showed that ACLSV-HBP had a similarity of 94.0% at nucleotide acid level and 96.4% at deduced amino acid level to SX/2. The recombinant plasrnid pGEX-KG-CP containing cp gene from ACLSV-HBP isolate was transformed into E. coli BL21 （ DE3 ）. A 46 kDa fusion protein was expressed under the induction of IPTG at 0.4 mmol/ L. Western-blot analysis showed that the cp gene was expressed effectively, and the fusion protein could give positive reaction with polyclonal antibody raised against ACLSV particles.

关 键 词：苹果褪绿叶斑病毒 原核表达载体 CP基因 分子生物学特性 SDS-PAGE分析 序列同源性 核苷酸序列 RT-PCR法 

分 类 号：S436.611.1[农业科学—农业昆虫与害虫防治]