沼泽红假单胞菌Rhp-PSP蛋白抑制TMV增殖的作用机理

The applicant previously identified an antiviral protein Rhp-PSP produced by a photosynthetic bacterium Rhodopseudomonas palustris. Rhp-PSP strongly inhibited TMV multiplication in plant, and attenuated infection ability of TMV-RNA by their direct contact in vitro. Based on the knowledge of reported nucleolytic activities among the Rhp-PSP homologues, the applicant hypothesizes that Rhp-PSP inhibits TMV multiplication through causing the degradation of TMV nucleic acids. Thus, our next research will focus on ① analysis of the binding and nucleolytic activity of Rhp-PSP on TMV nucleic acids in vitro through EMSA and Northern blot; ② detection of the TMV nucleic acids degradation by Rhp-PSP in vivo through plant transient expression of Rhp-PSP, TMV infectious clone and Northern blot; ③ comparing the antiviral efficacy between Rhp-PSP and its mutant lack of nucleolytic activity acquired through site-directed mutagenesis (SDM) technology. The results will verify the degradation of TMV nucleic acids afforded by Rhp-PSP, and subsequently help to reveal the inhibition mechanism of Rhp-PSP operating against TMV multiplication, and also providing theoretical information for the development of novel antiviral protein pesticides.

申请者前期从光合细菌沼泽红假单胞菌中分离到能抑制TMV增殖的抗病毒蛋白Rhp-PSP，并发现该蛋白可直接作用于TMV-RNA并破坏其侵染力。结合Rhp-PSP同源蛋白已报道的核酸降解功能，申请者推测Rhp-PSP通过降解病毒RNA抑制病毒增殖。因此，本项目拟：①采用EMSA、Northern blot方法，分析体外蛋白对TMV核酸的结合与降解作用；②构建蛋白植物瞬时表达与TMV侵染性克隆，并通过Northern与Western blot方法，分析植物体内蛋白对TMV核酸的降解作用；③采用SDM技术，获得缺失核酸降解功能的突变蛋白，并比较野生型与突变蛋白的抗病毒作用差异。研究结果将明确Rhp-PSP对病毒核酸的降解功能，阐明其抑制病毒增殖的作用机理，为开发新型蛋白质生物农药提供理论依据。

申请者前期从光合细菌沼泽红假单胞菌中分离到能抑制TMV增殖的抗病毒蛋白Rhp-PSP，并发现该蛋白可直接作用于TMV-RNA并破坏其侵染力。结合Rhp-PSP同源蛋白已报道的核酸降解功能，申请者推测Rhp-PSP通过降解病毒RNA抑制病毒增殖。本项目①采用了EMSA、Northern blot方法，分析体外蛋白对TMV核酸的结合与降解作用；②构建了蛋白植物瞬时表达与TMV侵染性克隆，并通过Northern与Western blot方法，分析了植物体内蛋白对TMV核酸的降解作用；③采用SDM技术，获得了缺失核酸降解功能的突变蛋白，并比较野生型与突变蛋白的抗病毒作用差异。研究结果得出：Rhp-PSP蛋白对核酸具有降解作用，且Rhp-PSP蛋白降解核酸的关键位点在C-端附近的β折叠上129位精氨酸上。Rhp-PSP蛋白通过Arg129位结合病毒RNA并参与其随后的降解，以实现对病毒增殖的抑制作用。其结果明确了Rhp-PSP对病毒核酸的降解功能，阐明了其抑制病毒增殖的作用机理，为开发新型蛋白质生物农药提供了理论依据。