沼泽红假单胞菌Atp2蛋白的原核表达及稻瘟病菌的互作蛋白初步筛选

Prokaryotic Expression of Atp2 Protein of Rhodopseudomonas palustris and Preliminary Screening of Interaction Protein in Magnaporthe oryzae

光合细菌沼泽红假单胞菌Rhodopseudomonas palustris PSB06是对稻瘟病具有良好防效的生防菌株。为进一步挖掘其生防代谢产物,前期通过蛋白层析分离技术,从PSB06培养液中鉴定到一个ATP合成亚基Atp2蛋白,ATP2基因编码序列为1431 bp。构建了pET32a(+)-ATP2重组表达载体,原核表达纯化后获得的Atp2蛋白产物用SDS-PAGE和Western blot进行鉴定。试验证明Atp2蛋白显著抑制稻瘟病菌的附着胞形成和在水稻寄主上的致病力。为阐明Atp2蛋白抑菌机理,通过酵母双杂交技术,以Atp2为诱饵筛选稻瘟病菌cDNA文库,获得了7个与Atp2有相互作用的蛋白质,分别是E3泛素连接酶复合物(MGG04978),两个核糖体蛋白(MGG16204、MGG04977)和4个假定蛋白(MGG09168、MGG01034、MGG03543、MGG00718),研究结果为揭示Atp2蛋白抑制稻瘟病菌的作用机制提供理论基础。

The earlier field experiment showed that PSB06(Rhodopseudomonas palustris PSB-06) had a good inhibition effect on rice blast. In order to further exploit the biocontrol products, an ATP synthetic subunit Atp2 protein was identified from PSB06 culture medium by protein Chromatography., with the coding gene sequence of 1431 bp. ATP2 genes were cloned from photosynthetic bacteria PSB06, and the recombinant plasmid pET32a(+)-ATP2 was constructed. The recombinant Atp2 was highly purified by Affinity Chromatography and analyzed by SDS-PAGE and Western blot. Atp2 protein could significantly inhibit the appressorium formation of Magnaporthe oryzae and the virulence on the rice host. In order to elucidate its antibacterial mechanism, yeast-Two-Hybrid-System was employed with Atp2 as a bait protein to screen the proteins interacting with Atp2 in the cDNA library of M. oryzae. Seven positive proteins were obtained, including E3 ubiquitin ligase complex(MGG04978), two ribosomal proteins(MGG16204, MGG04977) and four hypothetical proteins(MGG09168, MGG01034, MGG03543, MGG00718). The results provide theoretical basis for revealing the inhibition mechanism of Atp2 protein against M. oryzae.