P-loop,Bet v 1结构域的缺失及突变对GmPR10和Gly m 4l抑制大豆疫霉菌能力的影响

Effects of P-loop,Bet v 1 domain deletion and mutation on ability of GmPR10 and Gly m 4l to inhibit Phytophthora sojae

大豆疫霉根腐病是由大豆疫霉菌(Phytophthora sojae)引起的危害大豆生长的严重病害。课题组前期研究表明具有P-loop结构域的GmPR10(Gene Bank accession no.FJ960440)和具有P-loop、Bet v1结构域的Gly m 4l(Gene Bank accession no.HQ913577.1)抑制大豆疫霉菌生长,并且过表达GmPR10和Gly m 4l的转基因大豆植株可以提高对大豆疫霉根腐病的抗性。为研究GmPR10和Gly m 4l抑菌机理,本研究利用点突变技术,获得了GmPR10的P-loop结构域突变体(Gly48/Thr48和Gly^51/Arg^51)、Gly m 4l的P-loop结构域突变体(Gly^49/Ile^49和Lys^55/Pro^55)、GmPR10和Gly m 4l的P-loop结构域以及Gly m 4l的Bet v1结构域缺失突变体,并纯化回收相应突变体蛋白,进行体外抑制大豆疫霉菌试验。结果表明,突变或缺失P-loop,Bet v1结构域的GmPR10和Gly m 4l失去了抑制大豆疫霉菌(Race 1)生长的能力,说明P-loop、Bet v 1结构域对GmPR10和Gly m 4l行使抑菌功能至关重要。

Phytophthora root rot caused by the ommycete pathogen Phytophthora sojae is a serious disease affecting soybean growth.In our previous study,it showed that GmPR10 with P-loop domain and Gly m 4l with both p-loop and Bet v1 domains could inhibit the growth of Phytophthora sojae,and transgenic soybean plants of overexpressing GmPR10 and Gly m 4l could increase resistance to Phytophthora sojae.In order to further study the pathogen inhibition mechanism of GmPR10 and Gly m 4l,the P-loop structure domain mutant of GmPR10(Gly^48/Thr^48 and Gly^51/Arg^51),the P-loop structure domain mutant of Gly m 4l(Gly^49/Ile^49 and Lys^55/Pro^55),the P-loop structure domain deletion mutant of GmPR10 and Gly m 4l,the Bet v1 structure domain deletion mutant of Gly m 4l by using the point mutation technique were obtained and the purified proteins of corresponding mutations to inhibit Phytophthora sojae in vitro were carried out.The results demonstrated that GmPR10 and Gly m 4l with P-loop or Bet v1 domain mutation or deletion lost ability to inhibit the growth of Phytophthora sojae(Race 1),indicating that P-loop and Bet v1 domain were crucial for GmPR10 and Gly m 4l to perform anti-pathogen functions.