| Arthrobotrys oligospora is a filamentous fungi,which captures nematodes by producing trapping-device(three-dimensional network).Previous studies have found that amino acids or small peptides can induce A.oligospora to produce traps.Recently,our team also reported that urea can induce A.oligospora and other predatory nematode fungi to produce traps,indicating that amino acids and other nitrogen sources can regulate trap formation.In order to further understand the molecular mechanism of amino acid induced the trap formation,A.oligospora was used as the research strain,several proteins related to the regulation of amino acid signals were selected,including three proteins(AOL_s00043g 12,AOL_s00079gl87,AOL_s00097g622)related to the signal pathways of the rapamycin target gene(TOR,target of rapamycin),the general regulation of repressor protein kinase 2(Gcn2,AOL_s00215g639-18)and two G protein-coupled receptors(GPCR,AOL_s00054g626 and AOL_s00079g452),and their function were identified by gene knock out.Our results will help to investigate the mechanism of amino acid signal regulating the trap formation.The main experimental results:1.Construction of target genes(Tor,Gcn2 and Gpcr)knockout vector,transformation,and screening of positive transformants.The knockout vector of target genes were constructed by homologous substitution method and the knockout gene vectors were transformed into the protoplast of A.oligospora(AO)by Cacl2-PEG conversion method,we obtained positive transformants of six genes,such as ?12-27,?187-1,?187-5,?622-15,?639-18,?626-65 and ?452-94.2.Phenotypic comparison of wild type strain(WT)and mutant strains.The growth rate,stress resistance,sporulation ability and spore morphology of the transformants and WT were compared.The results showed that the growth rate of mutants and wild strain had no obviously difference on different media.Meanwhile,mutants had no showed significantly different from the WT in resistance to NaCl,SDS and H2O2,but A626-65 grew fast slightly than the WT in SDS plate.There were no significant differences in conidial morphology between the mutants and WT,but the number of conidia is different between the WT and mutants,the mutants ?626-65,?639-18,?187-5 and ?622-15 produced more conidia than the WT,and the?452-94 produced less conidia than the WT,these results indicated knocked out of nitrogen metabolism related genes does not affect the growth and the morphology of spores,but it have a certain effect on sporulation capacity in AO.3.Trap formation induced by nematodes,urea and amino acid.The main results include three aspects:1)Traps induced by nematodes,the mutants ?626-65 and ?639-18 produced more traps than the WT,and mutants ?72-27,?187-5,?622-15,?307-2 and ?452-94 produced less traps than the WT.2)Traps induced by 10 g/100 ml urea solution,the time of fungi produced traps was delayed.For example,the WT and mutants began to produce traps at 36 h,while the ?187-5 produced traps at 60 h,and lots of traps produced by all strains at 72 h.3)Traps induced by valine solutions(0.5 g/L,0.05 g/L,0.005 g/L),A.oligospora can produce traps in different concentrations of valine solution,while mutants but ?626-65 almost produced traps.With the increase of Val concentration,the A.oligospora and ?626-65 produced more traps,but the WT produced more traps than A626-65.Above results indicated that TOR and Gcn2 are closely related to the regulation of trap formation.Innovation in the study:1.Six genes(Tor,Gcn2 and Gpcr)of the AO genome were deleted,and the phenotypic characteristics of WT and mutants were compared,and their roles of these genes in the growth and development of AO were preliminarily estimated.2.Trap formation of the WT and mutants induced by valine.The WT and the Gpcr gene(AOL_s00054g626)knockout strain can produce traps induced by Val,while other mutants cannot produce traps under same condition,suggesting that the TOR and Gcn2 regulatory genes involved in regulation of trap formation. |
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