The Selection Of Chitinase From Arthrobotrys Sp.CX1 Strain And The Construction Of Its Engineered Bacteria

Plant pathogens can cause plant diseases,which have serious impacts on crop cultivation and crop yields,and are serious plant diseases worldwide.At present,chemical fungicides,heavy metals and traditional breeding methods are used to deal with plant pathogens.However,these have certain environmental pollution and adverse effects on crops.Therefore,it is necessary to research and develop more environmentally friendly methods for plant disease organisms.In the application of prevention.Chitinase can decompose fungal cell walls,inhibit fungal mycelial growth and kill host fungi by degrading the main component of plant pathogenic bacteria cell wall,chitin.At the same time,chitin zymolysis product chitin oligosaccharides have been reported to have antibacterial,antibacterial and other properties,and can regulate plant functions and promote plant growth and development.Therefore,chitinase is widely studied as a green and environmentally friendly plant insecticide.In this paper,a strain of nematode-feeding nematode Arthrobotrys sp.CX1 was selected as the research object,and five chitinase protein genes were screened in its genome and transcriptome database.According to bioinformatics analysis,it is known that the five chitinase proteins are all glycoside hydrolase 18 family proteins(named GH18 A,GH18B,GH18 C,GH18D,and GH18 E,respectively).The GH18 family protein is a chitinase.The family protein contains an inner barrel composed of 8 parallel ?-sheets and an outer barrel composed of 8 ?-helices(? / ?)8 barrel-shaped GH18 family The domain has two highly conserved active region sites: the chitin binding region highly conserved sequence SXGG and the enzyme active site highly conserved sequence DXXDXDXE.All five chitinase proteins contain GH18 family domains and two highly conserved active region sites.Five gene expression genes,GH18 A,GH18B,GH18 C,GH18D and GH18 E,were amplified from the c DNA of Arthrobotrys sp.CX1 strain by genetic engineering technology.In order to obtain efficient recombinant expression engineering bacteria,two expression hosts were selected to construct recombinant expression strains: prokaryotic expression host-E.coli BL21(DE3)p Lys S and eukaryotic expression host-Pichia pastoris X-33.E.coli BL21(DE3)p Lys S constructed recombinant chitinase engineering strain induced expression by IPTG,the protein size of the expressed target protein is the same as the theoretical value,the yield is large and it is in the form of inclusion body.The recombinantly expressed protein GH18 A was dissolved in 8 M urea and its inclusion body was renatured by concentration gradient to obtain 0.12 mg/ m L of target protein.With 1% colloidal chitin as the substrate,the chitinase activity was determined by DNS method,and no reducing sugar production was detected.The recombinant chitinase engineering strain constructed by Pichia pastoris X-33 was induced by methanol and expressed.The amount of recombinant protein expressed was less than 0.1 mg /m L in crude enzyme solution.Among them,GH18 A shows obvious bands,and its protein size is slightly larger than the theoretical value of 65.47 k Da,which may be due to the post-modification of the expressed protein by the eukaryotic host.GH18 B,GH18C,GH18 D,GH18E showed no obvious difference bands due to the low protein concentration.Using 1%colloidal chitin as the substrate,the enzyme activity of the chitinase was measured by DNS method.The measured enzyme activities of GH18 A,GH18B,GH18 C,GH18D,GH18 E were28.8,48.7,43.7,33.4,4.7 U / mg Enzyme.In summary,this paper successfully screened five chitinase protein genes derived from the nematode fungus Arthrobotrys sp.CX1,and constructed 10 recombinant engineering strains with two eukaryotic and prokaryotic expression hosts,and determined their In the eukaryotic expression host Pichia pastoris X-33 is the optimal expression recombinant host,four recombinant chitinases with higher activity are obtained.These research results provide more theoretical basis for the study of chitinase and lay a good preliminary work for the industrial production of chitinase.