PEG-CaCl_2介导苏云金杆菌CryⅠA(b)基因转化哈茨木霉

PEG-CaCl_2 mediated transformation of trichoderma harzianum with CryⅠA(b) gene from bacillus thuringiensis

为构建兼具杀虫防病双重功效的工程菌株，通过PEG-CaCl2介导的原生质体法将来自苏云金芽孢杆菌的CryIA（b）基因导入生防真菌哈茨木霉中．转化频率约为2～3个转化子／雌质粒DNA.Southern blot和RT-PCR检测结果表明，CryIA（b）基因已经整合到哈茨木霉基因组DNA中，并且在转录水平上得到了表达．各转化子都能够稳定遗传．对转化子的抑菌活性和杀虫活性检测结果表明，转化子不但保持了野生菌株的抑菌活性，而且表现出了一定的杀虫效果．转化子Tp1和Tp2对玉米螟幼虫的矫正死亡率分别为75．00％和72．75％．杀虫防病哈茨木霉工程菌株的构建为研究和开发新型微生物农药探索了新的诛径．

In order to construct a genetically engineered strain against insect pests and plant pathogens, CryIA（b） gene from Bacillus thuringiensis was transformed into Trichoderma harzianum using PEG -CaCl2 mediated transformation method with an efficiency of 2-3 transformants/μg plasmid DNA. The CryIA（b） gene used here was modified and synthesized from its wild gene according to plant codon bias without changing amino acid sequence. Putative transformants were analysised for the presence of CryIA（b） gene by Southern blot. RT-PCR analysis showed that the CryIA（b） gene was transcribed in T. harzianum transformants. Genetic stability of the transformants through mitotic cell division were also observed. Inhibition to plant Sclerotinia sclerotiorum indicated that no obvious difference in antifungal activities between the strains and wild strain, and meanwhile the engineered pathogen engineered strains had strong toxicity to insect pest Ostrinia furnacalis. The modified mortality of two transformants tested were 75.00% and 72. 73% respectively. The construction of the engineered strain should facilitate development of microbial insecticides against insect pests and plant pathogens.