Preliminary Study On The Functions Of Peroxisome-related Proteins Such As Arthrobotrys Oligospora Pex3p

Peroxisome is an important organelle,it contains many key enzymes of biochemical reactions.It participates in many important biological metabolism,such as hydrogen peroxide metabolism,fatty acid beta-metabolism and glyoxylic acid cycle.The peroxisome itself contains a variety of proteins,which are called Peroxins.The genes encoding these proteins are named PEX,and the corresponding proteins are Pexp.Peroxins are mainly composed of matrix proteins and membrane proteins.At present,Pex3p,Pexl6p and Pex19p are reported as membrane proteins of peroxisome,they are involved in the synthesis,transport and localization of peroxisome membrane proteins.Moreover,fatty acid degradation is a complex process in which peroxisomal beta-oxidation multifunctional protein(MFP)plays an important role and its coding gene is Mfp.Arthrobotrys oligospora is a typical fungus for studying the interaction between the fungi and nematodes,the fungus can capture nematodes by producing traps.In this study,three genes coding for Pexp including PEX3(AOL s00215g610),PEX16(AOL s00007g540)and PEtX19(AOL s00188g3),and two genes coding for Mfpl(AOL s00076g393)and Mfp2(AOL s00054g29),were selected to explore their roles in the growth and development,stress resistance,sporulation and nematode predation of A.oligospora.The main experimental results:1.Conserved domains,physicochemical properties and cluster analysis of peroxisome membrane proteins and MFPThe analysis of conserved domains confirmed that Pex3 protein contains the conserved domain of Peroxin-3(IPR006966),Pex16 protein contains the conserved domain of Pex16(IPR013919),and Pex19 protein contains the conserved domain of Pex19 protein(IPR006708).These Pexps contain different Pexp functional sites,but no common conserved domain was found.In the MFP protein family,both MFP proteins have HotDog superfamily domain(IPR029069)and MaoC-like dehydratase functional sites(IPR002539)involved in monoamine oxidase synthesis.MFP1 also contains a short-chain dehydrogenase reductase functional site(IPR002347)and NADP-binding superfamily domain(IPR036291).In addition,the molecular weight and isoelectric point of Pexp and MFP were predicted.Among the Pexp family,Pex3p has the largest molecular weight(MW),with 454 amino acids and 50.7 kDa MW,while the MWs of Pex16p and Pex19p are relatively small,and the isoelectric points of them are quite different.In the MFP family,the amino acids and MWs of MFP1 and MFP2 showed significant difference,with the number of amino acids being 901 and 313,and the MWs being 96.3 kDa and 34.3 kDa,respectively.The isoelectric points of MFP1 and MFP2 are similar,with 8.1 and 9.54,respectively.In cluster analysis,phylogenetic tree of Pexp contains three main branches,and Pex3,16 and 19 proteins are located on three different branches,respectively.Similarly,the phylogenetic tree of MFP contains two branches,MFP1 and MFP2 are also located in two different branches.2.Knockout of genes coding for PEX membrane proteins and MFPs,and related phenotypic characteristicsThree PEX membrane protein genes and two MFP genes were successfully knocked out by homologous recombination.More than two transformants were obtained from each gene,but Mfpl.After the deletion of PEX gene,no spores or traps were produced,and the nematocidal ability of the mutant was greatly reduced.Meanwhile,the vegetative growth of the mutants was slowed down,the mycelia became curved to varying degrees,and the septum was increased to a certain extent than that of the wild type(WT)strain.In addition,the utilization of fatty acids was also decreased in the mutants,and the resistance to antioxidant and cell wall perturbing agents of the mutants was different from that of the WT strain.In MFP knockout strains,?Mfp2 was decreased in fatty acid utilization ability to some extent,but there was no significant difference from the WT strain compared with the WT strain.In sporulation ability,sporulation number of the ?Mfp1 strains was decreased slightly,while sporulation number of the ?Mfp2 strains was increased slightly.Similarly,the spore germination rate of the ?Mfp1 strains had no significant difference from the WT strain,but the ?Mfp2 strains was lagged slightly behind the WT strain at 4 h and 8 h.Moreover,the nematocidal ability of the ?Mfp1 and ?Mfp1 mutants had no significant difference from the WT strain.The innovation of this paper1.Three PEX genes and two MFP genes of were successfully knocked out,and three PEX genes were found to be involved in the regulation of the growth,sporulation and trap foration in A.oligospora.