南京农业大学学报2013，Vol.36Issue(1)：6-12,7.DOI:10.7685/j.issn.1000-2030.2013.01.002
丁香假单胞大豆致病变种harpin编码基因的克隆表达与功能研究
Cloning, expressing and function of a harpin-encoding gene from Pseudomonas syringae pv.glycinea
摘要
Abstract
We amplified the hrpZ(hypersensitive response and pathogenicity)genes from Pseudomonas syringae pv.glycinea isolates Al and S1 genomic DNA by PCR technique, named hrpZPsgAI and hrpZPsgSI ,and the size were 1 026 bp and 1 038 bp. The fusion harpin protein was expressed in Escherichia coli BL21 and purified by GSTrap FF. The SDS-PAGE gel showed that fusion protein was 62×10 , and the molecular mass of harpinZPsgA1 and harpinZPsgSI were 35×l03. The concentration of pure harpinZpsgSl was 1. 1 mg·mL-1. Bioassay results showed that the protein was heat-stable and protein K sensitive, and was able to trigger hypersensitive response ( HR) in common tobacco. Besides, the HR elicitation of the protein in tobacco was dispelled by eukayotic metabolic inhibitors. Moreover,it could promote tobacco growth. Sequence alignment showed that hrpZ of Pseudomonas syringae pv. glycinea could be divided into two categories: a class with hrpZPsgAI as representative, including hrpZPsgl2, the other class with hrpZPsgSI as representative, including the hrpZ genes from the strains of rO and Race4. The nucleotide homology of these two classes was 79% and the protein homology was 77% , which contained abundant glycine(G) ,but had no cysteine(C). However, it did not show any sequence identity with those of other genus of gram-negative plant pathogenic bacteria. This study provides genetic resources for research and development of new microbial protein pesticides and for disease and pest resistance genetic engineering.关键词
丁香假单胞大豆致病变种/harpin编码基因/GST融合表达/烟草/过敏性反应/促生Key words
Pseudomonas syringae pv. glycinea/ harpin-encoding gene/ GST fusion expression/ tobacco/ hypersensitive response/ growth promotion分类
农业科技引用本文复制引用
张岩,伍辉军,周晓辉,高学文..丁香假单胞大豆致病变种harpin编码基因的克隆表达与功能研究[J].南京农业大学学报,2013,36(1):6-12,7.基金项目
国家863计划项目(2012AA101504) （2012AA101504）
国家公益性行业(农业)科研专项(201303015) （农业）
国家转基因生物新品种培育重大专项(2011ZX08004-004) （2011ZX08004-004）
国家自然科学基金项目(31100056) （31100056）
教育部高校博士点基金项目(20100097120011) （20100097120011）
国家博士后科研基金项目(2012M511770) （2012M511770）
