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By Maize Head Smut Ustilago Sporisorium axis (Sporisorium reilianum f.sp.Zeae) caused a serious threat to corn production systemic diseases, general infection at the seedling stage, heading stage performance typical symptoms, mainly against ear and male ear, once the disease is often no harvest the whole plant. This study aims to build PEG-mediated Sporisorium axis Ustilago protoplast transformation system, and attempting to clone the bacteria genome ras, gpd gene promoter for the development of a highly efficient Sporisorium axis Ustilago genetic transformation system and elucidate the pathogenic mechanism and so lay the foundation. The main results are as follows: 1, Ustilago Sporisorium axis protoplast formation and regeneration conditions were explored, tested strains isolated in our laboratory strains of black powder wire axis SR1, the effects of different cell age, different single enzyme as well as different combined into a single enzyme concentration enzyme solution after mixing efficiency of enzymatic discussed osmotic stabilizer, reaction temperature, reaction time and other effects protoplast formation and regeneration conditions, has been prepared Sporisorium axis Ustilago protoplasts The optimum conditions: SR1 3d activated after 1/20 cultured inoculum of about 24h, the spores were collected by centrifugation and washed once with sterile water, added with 0.5% of cleavage enzyme, 5% and 5% Driselase snailase three kinds of Enzyme Mix (enzyme solution in 1.0M / L sorbitol preparation) hydrolysis, placed in 28 ℃, speed 100r / m shaker and incubated 10min, protoplast yield up to 1.35 × 10 ~ 8 个 / mL, was rate reached 99.4 percent, the regeneration rate was 35.6%. 2, the plasmid DNA was Sporisorium PEG-mediated transformation of protoplasts Ustilago axis, the conversion efficiency of about 10 transformants / μgDNA. Clones stably expressing hygromycin B resistant transformant strain, subculture nine generations still hygromycin resistance. PCR analysis showed that exogenous DNA has been inserted into the wire axis Ustilago genome, with the wire transfer on behalf of Ustilago axes and stable inheritance. 3, clone Sporisorium axis Ustilago ras and gpd promoter to Sporisorium axis Ustilago genomic DNA as a template, according to the previously reported ras and gpd promoter related primers were designed using the PCR method of ras, gpd1 and gpd2 three promoter fragment. DNA sequencing analysis showed that after Blastn, ras sequences published online mushrooms ras promoter sequence has 98% homology, gpd with mushrooms gpd gene promoter sequence homology of 99%. Promoter 2.0 Prediction using software analysis found that ras, gpd2 a great possibility for the promoter, scores of 0.652,0.565. These fragments contain multiple core promoter sequences and TATA-box, CAAT-box and other cis-acting elements.
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