Procaryotic Expression Of Alternative Oxidase From Sclerotinia Sclerotiorum And Transformation Of BnWRKY40from Brassica Napus To Plants

Sclerotinia sclerotiorum is one of the most devastating plant pathogens, which can cause serious damage to both yield and quality of rapeseed. In order to better understand and control this pathogen, this paper made two aspects of research as follows.1. The alternative oxidase is a cyanide-resistant ubiquinol oxidase in fungi. In order to better elucidate the function of alternative oxidase of S. sclerotiorum, we expressed the alternative oxidase gene of this fungus in E. coli and prepared the antiserum of it. At first the3’-terminal350bp fragment of its alternative oxidase gene was amplified by PCR using cDNA from S. sclerotiorum as template. Then two copies of the fragment were linked and cloned into the prokaryotic vector pET-32a. The recombinant vector was transferred into BL21(DE3). SDS-PAGE result showed the fusion protein was expressed at a high level after induction by IPTG. The fusion protein was purified by IMAC affinity chromatography column and used to immunize rabbits to prepare antiserum. The titer of the obtained antiserum was showed above1:500000by ELISA. Western blotting result showed that the antiserum could specifically react with not only the fusion protein expessed in BL21(DE3), but also the alternative oxidase from S. sclerotiorum. The antiserum can be useful for studying the biological functions of alternative oxidase of S. sclerotiorum.2. WRKYs are a kind of transcription factor. Previous studies have demonstrated that WRKYs play essential roles in the responses of many plants to pathogens, but little is known about them in rapeseed. BnWRKY40was a novel WRKY gene cloned from rapeseed, which was found to be highly induced at48h after inoculation with S. scleroliorum. In order to better understand the function of BnWRKY40in the response of plants to S. scleroliorum, we cloned its coding region in both sense and antisense directions into a plant binary vector pBin438, and transformed tobacco leaf discs and rapeseed cotyledons by Agrohacterium-mediated transformation method. Some sense-transformed rapeseed and antisense-transformed tabacco plants have been acquired.