Functional Analysis Of Gene CuAO Related To Pathogenic Processes In Metarhizium Anisopliae

Metarhizium anisopliae is playing an important role in biological control of pests of an important class of insect pathogenic fungi. Compared with chemical pesticides, insects, fungal pathogens developed lasting insecticide action time, no environmental pollution, safety of non-target organisms, but there are also a serious impediment to the fungus the following shortcomings of the wide application of pesticides: death for a long time to kill insect low efficiency, control effect of instability, to address this issue, reveal the insects at the molecular level is particularly important pathogenic mechanism of pathogenic fungi. Metarhizium anisopliae infect a host insect, the spores secrete various proteinases degrade insect cuticle, and also in the formation of surface appressorium germination, appressorium formation and differentiation in the infection process plays an important role. Metarhizium anisopliae send through the main wall of the first invasion in vivo, in the hemolymph colonization, plunder the host by secreting toxins and nutrients to the host death, ultimately determines the outcome of infection is successful colonization of spores in vivo situation. During attachment spore formation and colonization stage in hemolymph specific expression characteristics of genes and gene expression, insect pathogenic fungi for the study of important pathogenic mechanism.Zhang Chuan-bo, etc. in the use of suppression subtractive hybridization (SSH) was constructed CQMa102 Metarhizium anisopliae in the pathogenic genes specifically expressed during the subtractive basis, we selected the number for the S-14 of Metarhizium anisopliae CQMa102 pathogenesis of high expression of EST sequences, the EST sequence length was 448bp, respectively, of the gene and the Penicillium CuAO (Peroxisomal copper amine oxidase) gene with high homology, the use of the laboratory building The Metarhizium anisopliae CQMa102 genomic DNA library, the success obtained CuAO gene DNA length; the use of RNAi techniques to study the CuAO function. The major findings are as follows:â‘ Constructed in our lab using Metarhizium anisopliae CQMa102 genomic DNA library, by homology comparison, for the CuAO gene DNA length. DNA sequence and cDNA sequences will be used bl2seq software comparison, the results showed that: CuAO gene DNA length 2118bp, containing an intron, one intron (114bp-191bp) boundary for the GG .... AC.â‘¡Using the method of RNAi gene function studies CuAO. CuAO gene fragment and RNA interference interference vector (pbar-RNAi-gpd-trp) connected, transformed into E. coli, and then particle bombardment transformation of Metarhizium anisopliae CQMa102, after PCR screening, were two CuAO RNAi strain.â‘¢Analysis RNAi strain of the number of 35 genes. The amount of attached spores when the spores growth on locust hind wing , the RNAi strain's attached spores to only a few, while the remaining spores are a lot of crawl-like hyphae . Wild type produce attached spores to the quantity of RNAi strain of 12.71 times.â‘£I have measure the virulence of RNAi strain and Wild type. The RNAi strain and wild type of lethal time were significantly different. The toxicity of RNAi strain has been reduce, suggesting that gene of Metarhizium anisopliae CQMa102 CuAO effects the virulence.