Preliminary Study On The Relationship Between Degradation Of Beauveria Bassiana And DNA Methylation

Beauveria bassiana, as one of the important entomopathogenic fungi, has been widely used for fungal insecticide development either in China or outside China. Due to its environmental compatibility, difficult to produce drug-resistant, abundant resources, lower development costs, etc., B. bassiana has been as an important factor to control the pest outbreak. Just as other fungal pesticides, the laboratory research and fermentation of B. bassiana has been limited, because of strain degeneration. These drawbacks hinder the utilization and industrial production of B. bassiana.UNA methylation exists widely in biological process of different organisms. Many organisms could emerge different traits by regulateon of gene expression with DNA methylation in the process of growth and development. This study will establish the foundation for further investigating the functional mechanism of DNA methylation in growth development of B. bassiana. And it is also benefit for industrial production and large-scale application of B. bassiana.The full-length cDNA of DNA methyltransferase gene Dim-2 was cloned from B. bassiana using SMART RACE RT-PCR. The sequence was also analyzed by bioinformatics methods. For multiple alignments and phylogenetic analysis, the deduced amino acid sequence of DIM-2 and other homologous sequences obtained from the GenBank database were analyzed using program ClustalX and program MEGA 4.0. And Realtime-PCR and HPLC methods were used to investigate the expression level of Dim-2 gene and the degree of DNA methylation in B. bassiana.The Dim-2 full-length cDNA sequence with 4021bp bp was deduced by sequence alignment and comparison with the 5’and 3’fragments. Except for 5’-UTR (untranslated regions) with 283 bp and 3’-UTR with 303 bp, the full-length cDNA of Bbtpsl possessed a 3429 bp open reading frame (ORF). The sequence bioinformatics analysis shows that the deduced BbDIM-2 protein had 1142 amino acids with calculated pi value of 6.13 and a molecular weight of 128 kDa. The CCD program of NCBI predicted that the deduced BbDIM-2 protein contain a active region of 5-mC ccatalytic synthesiscatalytic synthesiscccatalytic synthesis and a BAH domain.Multiple sequence alignment analysis of the BbDIM-2 showed a high homology with DNA methyltransferase protein of H Cordyceps militaris, Verticillium alboatrum, Neurospora crassa, Paracoccidioides brasiliensis and so on. Phylogenetic analysis based on twelve amino acid sequences of DIM-2 from fungi showed that these sequences were divided into two large groups. Sequence of BbDIM-2 was clustered into the subgroup with C. militaris. Furthermore, our phylogenetic analysis indicated that the homology of DIM-2 protein sequences between B. bassiana and C. militaris is 63%. In fact, both B. bassiana and C. militaris are entomogenous fungi and belong to the genus Cordyceps sensu lato,Colony morphology analysis of B. bassiana with subculture showed that the colonies appeared irregular after the 4th generation. And the sporulation was also reduced. When B. bassiana was subcultured for 10 generations, the mycelial growth became exuberant and sporulation significantly reduced. When B.bassiana was subcultured for 25 generations, the colonies was completely degraded, with mycelium soaring and few sporulation.The Realtime-PCR and HPLC analysis indicated that the degree of DNA methylation was changed in B. bassiana growth development, and there was a correlation between DNA methylation and expression of Dim-2 gene. The result also showed that both of the expression level of Dim-2 gene and the degree of DNA methylation reached the minimum in initial stage of sporulation. It was so inferred that there was an internal relationship between DNA methylation caused by Dim-2 gene and growth development in B. bassiana. Meanwhile, the experiment also showed that tussah rejuvenation can be used to reduce the degradation of methyl transferase Dim-2 expression, indicating rejuvenation may make bassiana to degradation.