The Gene Cloning And Prokaryotic Expression Of Antiviral Protein Rhp-PSP From Rhodopseudomonas Palustris PSB06

Tobacco mosaic virus (TMV), an important pathogen of crop diseases, is severely damaging economic crops and causing huge economic losses. In recent years, due to the chemical control of the economic crop disease will lead to the human health problems and result in the major environmental pollution, people advocate to use the non-toxic and harmless product to control the crop diseases. Protein pesticide, a new biological pesticide, is harmless for human, livestock and environment. It not only can increase the added value of agricultural products, but also can increase the economic benefits, so it has broad market prospects and competitiveness.Photosynthetic bacteria (PSB), a group of beneficial bacteria, can be used in agriculture as the organic fertilizer or the product of pesticide residue degradation. We found that PSB have control effect on Pepper Virus disease, but the mechanism has never been reported in literature.This paper is the first report on a new inhibitor that may be involved in the TMV of antagonization of Photosynthetic bacteria PSB06 strain. The protein was designated as Rhp-PSP. The perchloric acid-soluble protein (PSP) is reported as an endoribonuclease which can inhibit cell-free protein synthesis in rabbit reticulocyte lysate system. According to reports, Most PSP are found in animal, but rarely reported in microbial. In order to elucidate the molecular mechanism in the interaction process of Rhp-PSP and TMV, this paper focused on gene cloning by the prokaryotic expression system, for the purpose of protein expression, which lay the foundation for the next study. The results are followed by:(1) Through the activation of photosynthetic bacteria PSB06 strain, the extraction of PSB06 total DNA, and the sequencing of 16S rDNA fragment amplified by PCR for the determination of the strains. The result shows that the test strain is PSB06 strain.(2) Through the screening of the highly specific PSP primers, the PCR amplification of Rhp-PSP gene, the expression of Rhp-PSP gene in T5 expression vector, and the sequencing analysis were completed, and the result shows that the fragment sequence Rhp-PSP was perfectly and accurately expressed.(3) Through the expression of Rhp-PSP gene in E1 expression vector, the transformation in Rosetta competent cells with IPTG inducer, the SDS-PAGE were completed, the results show that the target gene Rhp-PSP was successfully expressed.