Construstion And Identification Of Mad1 Transgenic Beauveria Bassiana Strains

In the green pollution-free agriculture, microbial pesticide has been paid more and more attention and rapid development, although it has wide host range, with strong pathogenicity, harmless to humans and animals and other advantages, but the disadvantages are also obviously, such as pathogenic long latency, poor timeliness, high environment requirements, its promotion is still limited. Therefore to gain genetic transformation strains of insecticidal fungi by genetic engineering techniques, with higher insecticidal activity and adapt to pesticide market demand have important application value. The entomopathogenic fungus Beauveria bassiana played an important role in the biological control of pests. At present, a lot of transgenetic study of B. bassiana is mainly focus on the virulence related genes and direct conversion, such as protease gene, chitinase gene, and toxin genes, howerver, there are biological security problems. The adhesion protein gene from Metarhizium anisopliae used in this research, is not a directly toxic gene, but increasing the spore adhesion of entomogenous fungi, on insect body wall, thereby promote the virulence, shorten the time, to provide genetic engineering strains of B. bassiana for future breeding.Focal adhesion protein gene Mad1 was cloned from M. anisopliae, to construct genetic transformation vector. Protoplast of B. bassiana was prepared, and its concentration was 108sp/ml, and the regeneration rate was 15%. PEG-medium transformation of protoplast was used with selectable marker against coboxin, to obtained the stable genetic engineering strains gained after subculture and molecular identification.Adherence assay on insect and plant surfaces was used to detect the bioactivity of genetic strains. The results showed that there was no significant difference between adherent on onion epidermis of both strains, while it showed significant difference between adherent on locust wing cuticle, the adherent ability of genetic strain was higher than that of the later obviously. It indicated that the Mad1 gene gave an enhanced adherence on pest cuticle, but not on plant cuticle. The bioassay against the larves of Ostrinia furnacalis Guene showed that the genetic strain exhibit a higher toxicity than that of wild strain. The corrected mortality against the pests were 82.88% and 69.27% of the genetic strain and wild strain,respectively,and the LT50(3.278±0.346 days)was significantly shortened than the wild type(4.972±0.147 days), respectively, at 7th day after inoculation, it indicated that the insecticidal period of genetic strains was shorter than that of the wild strain obviously.