梨火疫病菌Sec依赖分泌蛋白EAMY_3046的鉴定及其原核表达  

Identification and prokaryotic expression of an Sec path protein EAMY_3046 of Erwinia amylovora

作　　者：严海璘 朱宗财 张王斌[1] 杜培秀 张超[2] 赵文军[3] 李为民[2] Yan Hailin;Zhu Zongcai;Zhang Wangbin;Du Peixiu;Zhang Chao;Zhao Wenjun;Li Weimin(College of plant science,Tarim University,Alar 843300,China;Biotechnology Research Institute,Chinese Academy of Agricultural Sciences;Chinese Academy of Inspection and Quarantine)

机构地区：[1]塔里木大学植物科学学院,新疆阿拉尔843300 [2]中国农业科学院生物技术研究所 [3]中国检验检疫科学研究院

出　　处：《植物检疫》2020年第4期1-6,共6页Plant Quarantine

基　　金：新疆生产建设兵团重点领域科技攻关计划项目(2018AB038)。

摘　　要：梨火疫病菌(Erwinia amylovora)是我国进境植物检疫性有害生物,针对其开展病原生物学研究,以及建立准确、快速、灵敏的检测方法至关重要。本研究从梨火疫菌株DSM 17948克隆了EAMY3046蛋白的编码基因Ea3046。该基因全长660 bp,编码220个氨基酸(amino acid,AA)。利用生物信息学与大肠杆菌碱性磷酸酯酶(alkaline phosphatase,PhoA)检测体系,明确EAMY3046蛋白具有Sec依赖分泌特性,其N-端17个氨基酸为信号肽(signal peptide,SP),C-端为成熟蛋白,包含203个氨基酸,命名为mEa3046。将DSM 17948菌悬液接种梨幼果,分别在0、8、24 h取样,提取总RNA,进行实时荧光定量PCR分析,结果显示Ea3046在梨火疫病菌侵染寄主植物过程中表达量显著上升,在8 h和24 h分别上调2.27倍和1.68倍。此外,构建原核表达载体pET-mEa3046,导入大肠杆菌BL21,经IPTG诱导16 h后,mEa3046在15℃条件下的表达量高于其在37℃的表达量,且蛋白以包涵体形式存在;利用Ni-IDA亲和层析纯化目标蛋白,经SDS-PAGE分析表明m Ea3046蛋白纯化成功。上述研究为EAMY3046抗体制备奠定了基础。Erwinia amylovora is plant quarantine pest of China,thus it is very important to study on pathogen biology and establish the accurate,fast and sensitive diagnosis methods. Herein,Ea3046,the coding gene of EAMY3046 was cloned from the E. amylovora strain DSM 17948. Sequence analysis disclosed that Ea3046 has a total length of 660 bp and encodes 220 amino acids(AA). Using bioinformatics analysis and Escherichia coli-based alkaline phosphatase(PhoA) gene fusion system,we determined that EAMY3046 was a Sec-dependent secretion protein,and its N-terminal 17-AA was a signal peptide(SP)and C-terminal 203-AA was the mature protein named mEa3046. The cell suspension of DSM 17948 was inoculated into the young fruits of pear,the pear tissues were collected at 0,8 and 24 hpi(hours post inoculation),respectively,and used for the total RNA extraction. Real time fluorescence quantitative PCR(RT-qPCR) showed that the expression level of Ea3046 was 2.27-and 1.68-fold higher at 8 hpi and 24 hpi,respectively when compared with that at 0 hpi. In addition,we constructed the prokaryotic expression vector p ET-mEa3046,and subsequently transformed into Escherichia coli BL21. After induction with IPTG for 16 hours,the recombinant expression of mEa3046 at 15℃ was higher than that at 37℃,and present as inclusion bodies. The expressed m Ea3046 was further purified by Ni-IDA affinity chromatography,and SDS-PAGE analysis showed that the target protein was successfully purified. The study laid a foundation for producing antibodies of EAMY3046.

关 键 词：梨火疫病菌 EAMY3046 Sec依赖分泌蛋白 原核表达 

分 类 号：S412[农业科学—植物保护]