Cloning Beauveria Bassiana Differentially Expressed Genes Induced By The Cuticle Of Cicadae And Silkworm Haemolymph Using MRNA Differential Display Technique

With the deepening of awareness for environmental protection, the problem of3R arising from chemical pesticides has received increasing attention. Using of biological insecticides to control insect pests has become a trend in the future. Due to the advantage of environmental compatibility, difficult to produce drug-resistant, abundant resources, lower development costs, etc., entomogenous fungi as an important lethal factor of the insects in agriculture and forestry ecosystems, have been as an important factor to control the pest outbreak. However, there are still many shortcomings, such as efficacy slow, low stress resistance for environment, therefore, how to increase the virulence and stress resistance of B. bassiana has always been the focus of research in the industry.To transform and build highly toxic, quick, stable new B. bassiana strains, we should understand the infection mechanism of B. bassiana. In this study, analysis of molecular strategy of infection process by simulating the infection process in B. bassiana was carried out, and trying to find a number of genes related to the infection process.Firstly, total RNAs were extracted from the mycelia of B. bassiana after24h induction in a liquid culture, which add to the cuticle of cicadae and silkworm haemolymph for induction, respectively, and then reverse transcription into cDNA using reverse transcription kit. The product added to the differential display primer system for PCR amplification after reverse transcription. The differential bands were recycled after the PAGE electrophoresis, and then the fragment of sequence were cloned and sequenced. The results showed that there were9genes related to the infection process,27gene sequences of putative proteins, and5unknown sequences. There were some genes related to the infection process, such as mature protease factor, allantoic permease, membrane G protein, second messenger cAMP, there was a significant differential expression of these genes in the cuticle of cicadae and silkworm haemolymph induced group compared to the control group.Secondly, the further analysis were conducted to verify the expression of protease maturation factor by real-time PCR technology, the results showed that there was a significant upregulation in gene expression(about eight times) in the cuticle of cicadae induced group compared to the control group.In this study, some genes related to the process of infection were identified by the mRNA difference display technology; furthermore, the differential expressions of the genes were analyzed by real-time PCR. These findings are expected to provide a basis for the research and elucidation of infection mechanism in Beauveria bassiana.