TasA抑菌蛋白对烟草黑胫病的防治效果研究

Biocontrol Effects of Antifungal Protein TasA on Tobacco Black Shank

【目的】烟草黑胫病是烟草疫霉菌侵染形成的一种严重危害烟草根茎的土传性疾病,给全球烟草种植业造成严重的经济损失。研究通过构建TasA蛋白原核表达重组菌,研究TasA对烟草黑胫病的抑制效果;并通过改变TasA蛋白的异源表达水平,以期提高该蛋白对烟草黑胫病的抑制效果。【方法】通过PCR扩增技术获得枯草芽孢杆菌抑菌蛋白TasA基因,构建大肠杆菌重组菌;并以烟草疫霉菌(Phytophthoranicotianae)为指示菌,研究TasA蛋白对烟草黑胫病的抑制效果。此外,通过改变克隆载体的拷贝数,研究TasA蛋白的表达量对其抑菌作用的影响。【结果】成功构建了三株大肠杆菌重组菌(pACYC-TasA、pCDF-TasA及pT7473-TasA菌株),且TasA蛋白在三株重组菌中都成功进行了异源表达,分子量为35 ku左右。pACYC-TasA菌株、pCDF-TasA菌株及pT7473-TasA菌株对烟草疫霉菌菌丝体均有抑制效果,抑制率分别为14.85%、25.55%及13.45%。【结论】抑菌蛋白TasA对烟草黑胫病具有显著抑制效果,且过表达pCDF-TasA重组质粒的工程菌对烟草黑胫病抑制效果最佳。

[Objective]Tobacco black shank is a soil-borne disease caused by fugal pathogens of Phytophthora nicotianae,typically infecting the roots and stems of tobacco plants and causing serious economic losses to the global tobacco industries.Therefore,recombinant strains overexpressing TasA were constructed to reveal the inhibitory effect of TasA on tobacco black shank.Moreover,the heterologous expression level of TasA was altered to improve its inhibitory effects.[Method]The TasA gene of B.subtilis was PCR amplified and overexpressed in Escherichia coli BL21(DE3)strain.The inhibitory effect of TasA protein on tobacco black shank was studied by using P.nicotianae as a pathogen indicator.In addition,the inhibitory effect of TasA under different expression levels was also studied by cloning TasA gene into three vectors with different copy numbers.[Result]Three recombinant E.coli strains(pACYC-TasA,pCDF-TasA,and pT7473-TasA strain)were successfully constructed.SDS-PAGE results revealed that TasA protein was overexpressed in all recombinant strains and its molecular weight was around 35 ku.The mycelial inhibition rate of P.nicotianae in pACYC-TasA,pCDF-TasA,and pT7473-TasA strain was 14.85%,25.55%,and 13.45%,respectively.[Conclusion]TasA protein shows a significant inhibitory effect on tobacco black shank,and recombinant strains overexpressing recombinant plasmid of pCDF-TasA exhibit the best inhibitory effect on tobacco black shank.