丁香假单胞大豆致病变种不同HrpZ蛋白差异区域功能研究

Functional identification of the differential regions of HrpZ protein from Pseudomonas syringae pv. glycinea

[目的]丁香假单胞大豆致病变种(Pseudomonas syringae pv.glycinea)A1和S1菌株编码产生的Hrp Z蛋白差异序列主要集中在C端的3个区域,并且这2个蛋白诱导非寄主HR的能力也存在差异。本研究将探究Hrp Z蛋白这3个差异区域在烟草上诱导HR(hypersensitive response)和抑制烟草花叶病毒(TMV)中所起的作用。[方法]采用常规PCR及重叠PCR方法将这2个hrp Z基因的3个差异区域分别进行互换,构建了相应的重组表达载体,表达并纯化得到了6个重组蛋白,相对分子质量均在41×103左右。并检测了其诱导HR活性、诱导植物抗病性。[结果]检测结果表明:重组蛋白Hrp ZS(S2→A2)和Hrp ZA(A3→S3)诱导HR的能力相比亲本都有所增强,6个重组蛋白诱导烟草抗TMV的活性都明显强于亲本,其中,Hrp ZA(A3→S3)和Hrp ZS(S3→A3)的活性最强。[结论]此试验表明Hrp Z蛋白的这些差异区域(第7、8、9个α-螺旋)是过敏反应能力的主要调控区域,同时C-端第8、9个α-螺旋区域与诱导植物抗病性也显著相关。本研究为改造Hrp Z类harpin蛋白激发子,提高其诱导抗性及功能域研究提供了理论依据。

[Objectives]In our previous study,two Hrp Z proteins,Hrp ZPsg S1 and Hrp ZPsg A1,were identified from Pseudomonas syringae pv. glycinea A1 and S1 strains,and the HR elicitation activity of Hrp ZPsg S1 in tobacco was stronger than that of Hrp ZPsg A1. Comparative analysis of their amino sequences showed that three different regions mainly existed in C-terminal. In order to clarify the relationship of these differential regions of Hrp Z proteins and the ability in eliciting the HR and disease resistance,we constructed 6 recombinant proteins by respectively exchanging three differential regions between Hrp ZPsg S1 and Hrp ZPsg A1. [Methods]After expression and purification,6 recombinant proteins about 41 × 103 were got,and HR elicition ability and Tobacco Mosaic Virus（ TMV） disease resistance ability were detected. [Results]Hrp ZS（ S2→A2）and Hrp ZA（ A3→S3）could induce stronger HR in tobacco compared with the wild-type proteins,furthermore,all of 6 recombinant proteins could reduce the phenotype caused by inoculation with TMV better than wild-type proteins,and among them Hrp ZA（ A3→S3）and Hrp ZS（ S3→A3）showed the best effects. [Conclutions]These results showed that the 7,8,9 α-helices were the main regulatory regions of HR,and the 8,9 α-helices were also related to the disease resistance. This study provides the theoretical basis for molecular modification of Hrp Z harpin protein in the future.