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

采用PCR方法从丁香假单胞大豆致病变种(Pseudomonas syringae pv.glycinea)A1和S1菌株中分别克隆到大小为1 026和1 038 bp的hrpZ基因(hrpZPsgA1和hrpZPsgS1),对该基因进行了原核表达和功能研究。SDS-PAGE显示其表达产物为相对分子质量62×103的融合蛋白,harpinZPsgA1和harpinZPsgS1的相对分子质量约为35×103。harpinZPsgS1的粗提蛋白经GSTrap FF纯化后质量浓度可达1.1 mg.mL-1。生物活性检测表明,该蛋白对热稳定,对蛋白酶K敏感,可以在非寄主植物烟草上激发过敏反应,过敏反应可以被真核生物代谢抑制剂抑制,并且对烟草有明显的促生作用。序列比对发现,来自丁香假单胞大豆致病变种的hrpZ基因可分为2类,一类以hrpZPsgA1为代表,包括hrpZPsg12,另一类以hrpZPsgS1为代表,包括来自r0和Race4菌株的hrpZ基因。这2类hrpZ基因的核苷酸同源性为79%,氨基酸同源性为77%,均富含甘氨酸,不含半胱氨酸,与假单胞菌属以外的其他革兰氏阴性植物病原细菌harpin编码基因不存在相似性。

We amplified the hrpZ（hypersensitive response and pathogenicity）genes from Pseudomonas syringae pv.glycinea isolates A1 and S1 genomic DNA by PCR technique,named hrpZPsgA1 and hrpZPsgS1,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 62×103,and the molecular mass of harpinZPsgA1 and harpinZPsgS1 were 35×103.The concentration of pure harpinZPsgS1 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 hrpZPsgA1 as representative,including hrpZPsg12,the other class with hrpZPsgS1 as representative,including the hrpZ genes from the strains of r0 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.