丁香假单胞大豆致病变种两个III型效应因子的克隆及功能分析

为了研究Ⅲ型泌出效应因子在丁香假单胞大豆致病变种中的作用,利用反向PCR技术,首次从丁香假单胞大豆致病变种全基因组中克隆得到两个效应因子HopAB1和HopAF1基因的同源物,分别命名为HopAB1s和HopAF1s。生物信息学分析表明,HopAB1s基因全长是1 572 bp,编码523个氨基酸;HopAF1s基因全长是855 bp,编码284个氨基酸。即基因的登录号分别为JF826562和JF826563。保守功能区预测显示HopAB1s在N末端包含一个E3泛素连接酶功能区。将这2个基因克隆到PVX二元表达载体并转化农杆菌,利用农杆菌介导的瞬时侵染技术在本生烟中表达,发现2个效应因子均能抑制由鼠凋亡因子激发的细胞程序性死亡;将烟草疫霉接种在表达效应基因的区域,发现效应因子能促进烟草疫霉侵染烟草,因此本研究得到的两个效应因子是免疫抑制因子,为进一步研究该菌的致病机理奠定基础。

Cloning and functional analysis of two type III-secreted effectors in Pseudomonas syringae pv. glycinea

For studying the pathogenesis of the soybean pathogen Pseudomonas syringae pv. glycinea, two types of homologous genes of III-secreted effectors, named as HopAB1s and HopAF1s respectively, were first cloned from Pseudomonas syringae pv. glycinea by means of inverse PCR (iPCR) method. The ORF of HopAB1s gene contained 1 572 bp, encoding 523 amino acid residues. The ORF of HopAF1s gene contained 855 bp, encoding 283 amino acid residues. The two genes registered in GenBank with accession numbers JF826562 and JF826563. The N-terminus of HopAB1s was sufficient for E3 ubiquitin ligases functional domain. The two effectors were inserted into the binary PVX vector, trans-formed into Agrobactrium tumefaciens GV3101, and the Agrobacterium-mediated transient expression experiments confirmed that two effectors functioned to inhibit the ability of the pro-apoptotic protein Bax inducing PCD in plant. Furthermore, infection experiment results showed that the effectors can promote ability of Phylophthora nicotianae infecting tobacco (Nicotiana benthamiana). Two cloned genes both belong to suppressive effector, our research is lay a foundation for revealed the molecular pathogenesis of Pseudomonas syringae pv. glycinea.