Functional Analysis Of Homologous Gene Fmo Related To Growth, Development And Pathogenic Process In Metarhizium Acridum

Flavin monooxygenase FAD-binding (FMO) commonly found in the endoplasmicreticulum of mammalian tissues, and it can oxidize a wide range of sulfur andnitrogen-containing exogenous substances, the metabolic toxic or carcinogenic substances,however FMO function is not clear in filamentous fungi. The laboratory was found in theprevious study, a FMO homologous genes in Metarhizium anisopliae infect host high toexpress in the host body, maybe is related to virulence. Research the M. acridum FMOgene (Mafmo) function is not only to further clarify the pathogenic mechanisms of fungi,but also to provide a theoretical basis for the screening of new antifungal or highly virulentstrains. In this study, through gene knock-out technology build the knockout strains Mafmo,analyzed Mafmo gene on the M. acridum, growth, development and Resisting Stress.The main research contents are as follows:1. Bioinformatics analysis sequence of MafmoTo select EST sequence number of CJY1439that M. acridum express in thehost-specific gene built from the laboratory, and analysis of the sequence homology byNCBI Blastx. To search out CJY1439of genomic sequence correspond with nucleotideblasts and predicte amino acid sequence by the GENSCAN1.0and GeneMark.2. Obtained knockout strains of MafmoTo designe Primers according to the wild-type (WT) M. acridum Ma102genomicDNA and amplificate out Mafmo the left and right DNA fragment to insert of pK2-PBvectors Bar of5’ or3’ respectively, construct Mafmo knockout vector and transformatethrough Agrobacterium-mediated. By PCR analysis and Southern blotting method toIdentificate Mafmo gene knock out.3. Analysis function of Mafmo⑴The role of Mafmo in virulenceMafmo knockout strains or wild-type strain of fresh spores formulated into a sporesuspension were used in injection or the infection two ways to identify the role of Mafmoin virulence. By observe the death of the host time after two ways to deal with, analysis semi-lethal time for Mafmo gene knock out clearly.⑵The effect of Mafmo in infection process①The influence of Mafmo in the host blood chamber inoculated strainsIn vivo: Mafmo knockout strains or wild-type strain of fresh spores to prepare thespore suspension, and inject, take the host blood observed number of insect bacterial.In vitro: To observe spore germination and growth, take the fresh blood of the host (toremove or not to remove blood cells) were inoculated with fresh spores of Mafmoknockout strains or wild-type strain.②The fuction of Mafmo at primary infect stageSpore germination and morphology of appressorium: Mafmo knockout strains orwild-type strain of fresh spores to prepare to the spore suspension were inoculated hostwings, observe spore germination and the infection structure appressorium formation timeto analysis between whether there are differences.⑶The impact on stress resistance growth of MafmoIn the high osmotic pressure (0.4MKCl,1M sorbitol,1M mannitol), high-oxidant(30%H2O2), and cell wall interference agent (50μg/mL Calcofluor White,500μg/mLCongo Red,0.01%sodium dodecyl sulfonicsodium) or hypoxic conditions to cultureMafmo knockout strain or wild-type strain fresh spores preparation of the spore suspension,observe the differences in morphology, sporulation, biomass accumulation.The main results are as follows:1. The EST sequences is number CJY1439and length of710bp. The Blastx foundCJY1439is the homolog of monooxygenase FAD-binding (FMO) sequence and namedMafmo. The gene was predicted to encode193amino acids with a molecular weight of21.8kDa, pI of8.14and did not contain a signal peptide.2. By PCR analysis and Southern blotting method, we got four stable knockout strainsof Mafmo (△Mafmo1、△Mafmo3、△Mafmo5、△Mafmo6) at last.3. The result of Mafmo function⑴Biological virulence test results: after injection spore suspension, Mafmo knockoutstrains than the wild-type strain to result in the death of the host of semi-lethal timesignificantly earlier (the Mafmo knockout strains semi-lethal time:△Mafmo33.83±0.10d;△Mafmo53.59±0.13d;△Mafmo63.23±0.21d, wild-type strain Semi-lethal time:4.99±0.17d), after infection spore suspension, Mafmo knockout strains and the wild typestrain semilethal time is no significant difference. It indicated that Mafmo is related tovirulence. ⑵①vivo blood culture results display: Mafmo knockout strains or wild-type strainfresh spores to prepare the spore suspension by injection into the host’s body, take the hostblood observed that insect bacterial of Mafmo knockout strains than the wild-type strainnot only earlier and more, the difference in the number is significantly; vitro blood cultureresults display: blood cultured the wild-type strain and Mafmo knockout strains sporessuspension36h respectively. The difference is significant; blood for removal ofhemolymph cultured wild-type strain and Mafmo knockout strains respectively was nosignificant difference in the number of bacteria.②Mafmo knockout strains or wild-typestrain fresh spores preparation of the spore suspension were inoculated in the host wings20h, germination rate of the wild-type strain was significantly higher than Mafmo knockoutstrains; the appressorium relative formation rate was no significant difference. So theMafmo affect strain germination on host surface not the formation of appresorium.Above①,②show that: the Mafmo not only have active when strain into the hostblood chamber, also affected the growth and development of M. acridum at infection earlystage.⑶under high osmotic pressure, high oxidant interference agents and hypoxicconditions, culture knockout strains and the wild-type strain respectively, both showedsignificant differences in morphology, sporulation, biomass accumulation. The resultsshowed that the Mafmo impact on the growth and development of M. acridum under stressconditions.