Research On Cloning, Expression And Bioactivity Analysis Of The Chitinase Genes From Entomogenous Fungi

Since microbial pesticides have the advantage of ample raw materials, specificprevention, less resistant among treatment objects and environment friendly, theyhave become a pillar industry of the biological control. The Beauveria bassianafungus and Verticillium lecanii is the most widely used and the most study of insectpathogenic microorganisms. Chitinase is the main insecticidal active substances. Butsome pathogenic fungal cell walls also contain chitin, so these two kinds ofpathogenic microorganism chitinase also inhibited on pathogenic fungi. Cloning ofchitinase gene from Beauveria bassiana and Verticllium lecanii, these proteins wereexpressed in Escherichia coli, to study on the antibacterial activity. And becausethey are conpletely chitinase, the combination of the two fungis can be used as a newtype of biopesticide in pest control. The fusion gene can produce two differentchitinase protein and effectively control of disease resistance by fermentation at atime. So the results are as follows:1In this study, the primers were designed from the sequence information ofBeauveria bassiana and Verticllium lecanii, By the method of PCR, the chitinase wascloned from Beauveria bassiana and Verticllium lecanii. The Beauveria bassianachitinase gene has an open reading frame comprising of1047bp nucleotide sequences,encoding a protein of348amino acids. The molecular weight of the protein is384kD,its isoelectric point5.94. The Verticllium lecanii chitinase gene has an open readingframe comprising of1272bp nucleotide sequences, encoding a protein of423aminoacids, the molecular weight of the protein is46.7kD, BbCHI and VlCHI gene wereexpressed in Escherichia coli. and the expression protein reach a maximum at4h.2By overlapping PCR method,the Beauveria bassiana and Verticllium lecaniichitinase gene will constructed,and fusion gene will expressed in Escherichia coliBL21,fusion proteins was detected by SDS-PAGE electrophoresis, and found thatreached the maximum amount in the4h. 3By confront culture method, three kinds of protein buffer determined theinhibitory activity on Setosphaeriaturcica and Sclerotinia sclerotiorum. Inhibitoryeffect of different buffer concentration protein on the growth of Setosphaeriaturcicamycelium was determined. Inhibitory effect of different buffer concentration proteinof corn leaf spores was determined in the supravital maize leaves. Inhibitory effect ofdifferent buffer concentration protein on the growth of Sclerotinia sclerotiorummycelium was determined. Inhibitory effect of different buffer concentration proteinof Sclerotinia sclerotiorum mycelia was determined in vitro of tomato leaves. Theresults show that Beauveria bassiana chitinase protein of antibacterial activity was asmuch as Verticllium lecanii. But the antibacterial activity of fusion protein chitinasefusion protein is stronger than alone4Determination of antibacterial activity of some selected organic chemicalpesticide to Sclerotinia sclerotiorum and Setosphaeriaturcica. Research shows that thebest inhibitory effect on Sclerotinia sclerotiorum is Epoxiconazole, the best inhibitoryeffect on northern corn leaf blight is flusilazole and the best inhibitory effect on sporeof Setosphaeria turcica is prochloraz.The antibacterial activity of Prochloraz on twospecies of fungus are good. Therefore prochloraz is a kind of very goodbroad-spectrum fungicide, It is a guiding role for the future agricultural production.For the first time, Beauveria bassiana and Verticllium lecanii fusion gene wasexpressed in Escherichia coli BL21.It can reduces multiple fermentation cost, but alsoenhance their inhibitory activity. The first study showed that chitinase fusion gene notonly has insecticidal activity but also has bactericidal activity. The suppression ofplant diseases and insect pests of fusion protein and gene of chitinase provides a basicresearch method, It can provide valuable materials and ideas for the development ofbiological pesticide.