| All insects are easily infected by pathogenic fungus and a large area of insect colony is dead due to spreading and diffusion of pathogenic fungus,so it is very necessary for pathogenic fungus as an important biological-control resource to explain its molecular mechanism of infectious pathopoiesia.As an important structure outside of fungal cells,fungal cell wall,mainly composed of polysaccharide(glucan,chitin or mannan)and glycoprotein,can provide the protection for adverse external environment and host's immunoreactions.Glucan is a main composition of fungal cell wall and glucoside transferase plays an important role in functional performance of glucan due to hydrolysis and transferase activity and mainly impacts integrity of spore wall in saccharomyces cerevisiae and aspergillus fumigates.But its functional mechanism is still not clear during fungal growth and development.So,as for the subject,take locust metarhizium as test materials,adopt gene knockout technology to set up glucoside transferase knockout strain and reverse strain and carry out a primary analysis for growth,virulence and stress resistance.Main research contents:1.Bioinformatics analysis was used to analyze gene structure and function.2.Construct the knockout and restoration strains.The knockout and restoring strains were constructed by homologous recombination,and the correct transformants were obtained by PCR and Southern blotting.3.Analysis of gene function(1)growth characteristics analysis: scraping fresh ripe knockout,wild type,and the recovery strain spores formulated into a spore suspension.The germination rate and sporulation were observed.Under the microscope with a hemocytometer counting and statistical spore number,statistics and analysis of data inversion.The constant temperature of 28 DEG C in cultured 20 h in the box,stained with fluorescent whitening agent0.5mg/ml,observe the mycelium structure under the fluorescence microscope.(2)analysis of resistance characteristics: scraping fresh mature knockout,wild type,and reply to strain spores formulated into 1×107 /ml spore suspension were placed in 45 DEG C water bath in 1350 mW and m-2 under UV light treatment,under the microscope to compare the statistics of germination rate,germination half the inhibition time difference.(3)Virulence Analysis: scraping fresh ripe knockout,wild type strain spores prepared into spore suspensions,used in injection and surface infection infection of locust,every12 h a statistical mortality,until all the death,through statistical analysis of virulence of semi lethal time difference.Main findings:1.It is analyzed from bioinformatics that Mwg1: the accession number was EFY91318.1,cDNA is 1367 bp,the size of amino acid is 406 aa,isoelectric point is 5.22 pI and molecular weight is 41 kDa,including one intron;Mwg2: the accession number was EFY92943.1,cDNA is 998 bp,the size of amino acid is 277 aa,isoelectric point is 5.03 pI and molecular weight is 26 kDa,including one intron;Mwg3: the accession number was EFY86494.1,cDNA is 1669 bp,the size of amino acid is 511 aa,isoelectric point is 4.89 pI and molecular weight is 52 kDa,including two introns.2.The test set up ?Mwg2,?Mwg3,CP1,CP2 and CP3 strains as well as??Mwg1/Mwg2 and ??Mwg2/Mwg3 strains at the same time and obtained correct transformants by PCR and Southern blot.3.Influence of glucoside transferase gene on growth of locust metarhizium:(1)Germination and sporulation: as for ?Mwg1 and ?Mwg3,sporulation increases and germination advances compared to WT and CP;as for?Mwg2,sporulation decreases and germination delays compared to WT and CP;as for ??Mwg1/Mwg2 and??Mwg2/Mwg3,sporulation increases and germination advances compared to WT.(2)Hypha structure: as for ?Mwg1,hypha ends are longer and thinner and distance between hypha diaphragm is increased compared to WT and CP;as for ?Mwg2,branch number increases significantly compared to WT and CP;as for ?Mwg2,hypha ends are longer and thinner and distance between hypha diaphragm is increased compared to WT and CP;as for ??Mwg1/Mwg2 and ??Mwg2/Mwg3,hypha ends are longer and thinner and distance between hypha diaphragm is increased compared to WT.4.Influence of polyglucoside transferase gene on stress resistance of locust metarhizium:(1)UV resistance: as for ?Mwg1,?Mwg3,??Mwg1/Mwg2 and ??Mwg2/Mwg3,germination rate of spores advances and UV resistance strengthens after UV treatmentcompared to WT and CP;as for ?Mwg2,germination rate of spores delays and UV resistance weakens after UV treatment compared to WT and CP.(2)Wet and heat tolerance: as for ?Mwg1,germination rate of spores advances and wet and heat tolerance strengthens after wet and heat treatment compared to WT and CP;as for ?Mwg2,germination rate of spores delays and resistance to wet and heat tolerance weakens after wet and heat treatment compared to WT and CP;as for ?Mwg2 and??Mwg2/Mwg3,there is no significant difference compared to WT AND C;as for??Mwg1/Mwg2,germination rate of spores after wet and heat treatment advances compared to WT.(3)Sensitive agent of cell wall: on 1/4SADAY plate with a congo-red sensitive agent of cell wall,as for ?Mwg1,colonial morphology significantly reduces and it grows slowly compared to WT and CP;as for ??Mwg1/Mwg2,it grows slowly and other knockout strains do not have an significant influence compared to WT;on 1/4SADAY plate with NaCl and Sor,the growth situation of each strain is not influenced significant compared to WT,so it shows that glucoside transferase do not impact the tolerance of metarhizium to hyperosmosis.5.Influence of polyglucoside transferase gene on locust metarhizium virulence:Mainly adopt drop and injection(namely skin infection and in vivo injection)to respectively prepare the spore suspension of knockout and WT strains to infect asiatic migratory locust to analyze the influence of glucoside transferase gene to locust metarhizium virulence.Findings show that: in case of skin infection,virulence for ?Mwg1,?Mwg2 and ?Mwg3 is not impacted compared to WT;but virulence for ??Mwg1/Mwg2 and ??Mwg2/Mwg3 is significantly weakened compared to WT.In case of in vivo injection,the virulence has no difference between ?Mwg2 and WT;but virulence for?Mwg3 strengthens compared to WT;virulence for ??Mwg1/Mwg2 and ??Mwg2/Mwg3 strengthens compared to WT. |
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