绞股蓝抗TMV蛋白的分离及编码基因的序列分析  

Purification of a Novel Anti-TMV Protein from Gynostemma pentaphyllum and Sequence Analysis of Its Partial DNA Coding Region

作　　者：林毅[1] 陈国强[1] 吴祖建[1] 林奇英[1] 谢联辉[1] 

机构地区：[1]福建农林大学植物病毒研究所福建省植物病毒学重点实验室,福州350002

出　　处：《农业生物技术学报》2003年第4期365-369,共5页Journal of Agricultural Biotechnology

基　　金：福建省科技厅重点项目(No.98-Z-178;99-Z-193)

摘　　要：以半叶法测定抗烟草花叶病毒(Tobacco mosaic virus,TMV)活性为监控手段,采用(NH4)2SO4分级沉淀、阳离子交换及亲和层析等方法,从绞股蓝(Gynostemma pentaphyllum)中分离到一种抗TMV蛋白,其分子量约为27 kD,N端19个氨基酸残基为DINFSLAGADGQTYNTFIA(SWISS-PROT登录号为P83206).N端序列分析表明,它是一种新的核糖体失活蛋白(ribosome-inactivating proteins,RIPs),命名为gynostemmin.gynostemmin与其它植物RIPs的N端同源性介于10%～73%之间,其中Y1,17构成植物RIPs N端的'钉子'位点;gynostemmin与葫芦科植物RIPs的同源性为31%～73%,其中F4L6A9Y14F17I18 6个残基为葫芦科植物RIPs N端'钉子'位点.根据N端序列和葫芦科植物RIPs下游保守氨基酸序列设计简并引物,采用PCR扩增并克隆了编码gynostemmin的基因,包含609个碱基,推导出的203个氨基酸残基约占成熟蛋白的75%,软件分析表明,它含有核糖体失活蛋白的活性位点区.A novel anti-Tobacco mosaic virus(TMV) protein designated as gynostemmin was isolated from Gynostemma pentaphyllum. Its molecular mass was 27 kD and the N-terminal 19 amino acid residues were DINFSLAGADGQTYNTFIA(SWISS-PROT accession number P83206). Blast analysis revealed that gynostemmin might be a new type I ribosome-inactivating proteins(RIPs).The comparison of N-terminal amino acid sequences of gynostemmin and other RIPs showed that F4L6A9Y14F17I18 were completely conserved in RIPs from Cucurbitaceae and Y14F17 were completely conserved in RIPs from high plants. The partial DNA coding region of gynostemmin was amplified by PCR using two degenerated primers based on the N-terminal sequence and the highly conserved amino acid residues in the downstream of RIPs from Cucurbitaceae. And the amplified fragment was cloned and sequenced. The 203 amino acid residues deduced from the partial DNA coding region was predicted to possess Shiga/ricin ribosomal inactivating toxins active site, DNA binding motif, and four potential N-glycosylation sites.

关 键 词：绞股蓝 抗TMV蛋白 分离 编码基因 序列分析 抗烟草花叶病毒 

分 类 号：S567.237[农业科学—中草药栽培] Q943[农业科学—作物学]