Functional Analysis Of Mwg1Gene In Metarhizium Acridum

The fungal cell wall is an important organ of fungi, it plays an irreplaceable role in theprocess of protection fungal and against adverse external environment and fungal growthor development. Polysaccharides(Glucan, a few chitin, mannan) and glycoproteins aremain ingredients of the fungal cell wall. Currently, Study reported more the synthesis ofenzymes associated fungal cell wall, but reported less hydrolasesneed who need forreconstruction and expansion during cell growth. The glucanosyltransferase having ahydrolysis and transferase enzyme activity, it extend the1,3-beta-glucan-chain or branchedchains connected to form a1,6-β-glycosidic bond and1,3-beta-glucan chain in yeast.However, glucanase transferase biological function of the gene in fungal growth anddevelopment, infection, role in the resilience process is not yet clear. For clearunderstanding their function, the project Metarhizium acridum as experimental material,using of gene knockout technology to build glucan glycoside transferase Mwg1inknockout strains and biological virulence determination, determination of cell wallpolysaccharide and resistance phenotype observed, to analysis biological function of Mwg1.Main research contents:1Bioinformatics analysis of gene structure and function of the Mwg1.Search the Mwg1the genome sequence and amino acid sequences from GenBank; Onthe NCBI Blast analysis the Mwg1sequence of the sequence homology with other genes.2. Building Mwg1knockout strains.Primers were designed according to Mwg1the genome, from Metarhizium acridumMa102genomic DNA amplification out Mwg1left, right arm DNA fragments andrespectively inserted in the the pMaDisrupter-3carrier Bar gene5’end or3`end to buildMwg1knockout vector. By Agrobacterium mediated transformation get Mwg1strains.By PCR analysis and Southern blotting method identification Mwg1strains.3.Mwg1gene function analysis.1) Mwg1role in the the Metarhizium resilience growth: In PDA culture medium andadded CR, SDS, CFW cell wall interference agent and KCl, sorbitol, mannitol penetrant toculture Mwg1with WT as control, Observe the colony morphology, mycelium differencein sporulation, biomass accumulation; Coating Mwg1and WT spores on PDA medium,irradiated with UV1-5h at28°C for24hours, statistical analysis whether differences exist between the relative germination rate; The Mwg1and WT spores with a45°C water bathincubator incubated for1-5h or65°C incubator placed2-8h the coating on PDA mediumand incubated at28°C for24hours, statistical analysis of the relative germination rate, andwhether there are differences between WT and Mwg1;2) Cell wall polysaccharide content: mycelium cell wall of Mwg1and WT afteracid treated, then with3,5-DNS colorimetric determination of total sugar content andNaCl-H3BO3solution UV spectrophotometric colorimetric determination of mannancon-tent and SLP colorimetry determination of β-1,3-glucan content to analysis of changes inthe composition of the cell wall polysaccharides.3) The Mwg1strain virulence determination. Use Mwg1and WT spore suspen-sion in vivo injection and body surface infection are two ways infection oriental migratorylocust, Observe the determination of in vivo injection or surface infection time of locustsdeath, through the Statistical half time of death to analysis Mwg1and WT strain with hostof locusts virulence differences.The main findings:1. Bioinformatics analysis results display Mwg1genes have an intron and composedof406amino acids, its protein molecular weight of41kD, isoelectric point to5.22;BLAST analysis found that the gene and other fungal cell wall transferase gene there is ahigh homology.2. Successfully constructed the knockout vector Mwg1; PCR analysis and Southernblotting was used to verify the two Mwg1strains.3.Analysis of gene Mwg1function1) On PDA containing Congo Red medium, Mwg1significantly slow growth,colony diameter is too small, microscopic observation found Mwg1hyphae significantlyreduced compared with WT; PDA medium containing such as KCl penetrants, by Mwg1and WT growth uniformly suppressed, but no significant differences between the two;fluorescence microscopy mycelium of Mwg1and WT found significantly reducedMwg1than WT hyphae branch, a significant increase in the compartment length; OnPDA medium was added SDS or CR, Mwg1than WT sporulation decreased significantly,spore germination rate difference was not significant; by ultraviolet irradiation2-4h,Mwg1spore germination was significantly higher than that of WT; after45°C water bathor65°C after dry heat treatment, Mwg1spore germination was no significant differencewith WT wild-type.2) Mycelium cell wall sugar, mannan, glucan measurement results: Mwg1compared to WT, total sugar and β-1,3-glucan significantly reduced, but a significant increase inthe content of mannan.3) Mwg1strains with the WT strain virulence test results show that:In vivo injection or surface infection process, Mwg1and WT semilethal time nosignificant difference.Main conclusions:Mwg1influences the formation of cell wall structure in metarhizium acridum. Lackingof Mwg1can cause ratio change of β-1,3-glucan and mannan in cell wall, reduce separa-ed mycelium and make internodes longer, however, have no impact on the virulence of metarhizium acridum. The increase of ratio of1,3glucan and mannan in cell wall may enha-nce the UV resistance of metarhizium acridum.