Construction Of CDNA Library Of Alternaria Sp., Cloning And Fusion Expression Of Activator Gene Of Alternaria Sp.

Activator is a series of novel protein,which is isolated from fungi. Activator had a high insecticidal activity to many kinds of virus disease, for example TMV and CMV,moreover,it can greatly improve crop 's tolerance to drought and increase crop yield. The construction of the genetic engineering bacteria based on identify activator gene is a basic content of the research on the mechanism, structure and function domain.A cDNA entry library and a cDNA expression library of Alternaria sp. were constructed with a new directional cDNA library construction method using an in vitro site-specific recombination reaction, based on the integrase-excisionase system of bacteriophage A. Have a research on expression of activator protein and purity of target protein. it provide usable materials and tools for the research of molecular biology on the genetic background of Alternaria sp., These study lay a foundation for exploreing the structure and function of plant activator.1. The entry library has a high titer of 6×10~6 and a total clones of 5.6×10~7,in wich 100% clones are recombinant and the size of average insert cDNAs is 1.53Kb. In order to screen library by Activator antiserum, we perform the LR recombination reaction to transfer our entry library into Gateway(?) vector to creat expression library. The expression library has a high titer of 1.53 × 10~6and a total clones of 6.2 × 10~6,in wich 100% clones are recombinants and the size of average insert cDNAs is 1.62Kb.2. A full-length coding sequence of 882bp that contains initiation codon and termination codon was obtained through immunological screening of the expression library with the antiserum of anti-Alternaria sp. Protein. it was found that the protein sequence was similar to mass spectrum analysis result of 35kD protein of plant activator.The search results of NCBI BlastN and BlastP showed that no sequence with identities of more than 40% was found, so that it's a new protein.3. The gene of 32kD protein was subcloned into pET-21b vector which has T7 promoter and His·Tag sequence. The fusion protein was attempted to express in E.coli BL21 and purified it by affinity chromatography.