The Proteomic Study Of Soil Inhibiting Spore Germination Of Arthrobotrys Oligosporum And The Molecular Mechanism Of Benzaldehyde And Ammonia Inhibiting Spore Germination

Plant-parasitic nematodes cause severe damage to agriculture in our country,Biocontrol fungi are important nematode biocontrol resources.However,either germination or growth of fungi are inhibited by Soil fungistasis.As a result,fungal biocontrol agents have not reached a reliable and consistent level.In order to study the molecular mechanism of fungal spore germination inhibited by soil fungistasis,firstly,we use iTRAQ-LC-MS/MS proteomics technology studied the protein group of spores that were suppressed by soil suspension and analyzed the molecular mechanism of soil suspension on the spore germination of Arthrobotrys oligospora.In the previous study,six major proteins related to spore germination were screened out from the data of the differentially expressed proteins which respectively inhibited by benzaldehyde and ammonia,the volatile substances from soil environments.The coding genes of six proteins?AOL_s00169g229,AOL_s00215g 160,AOL_s00075g181,AOL_s00083g51,AOL_s00075g106,AOL_s00215g7?were knocked out by homologous recombination.The growth rate,sporulation conditions,spore germination rate and stress resistance of mutant strains were studied.This study has made some useful explorations on the molecular mechanism of the soil inhibit the germination of the spore.The main results were described as follows:1 Base on the iTRAQ-LC-MS/MS analysis and the MaxQuent search results,The expression of up regulated and down regulated proteins was screened by 2 times and 0.5 times.The results show:ungerminated conidia proteins as a control,211 proteins were up-regulated and 102were down-regulated in the normal 24h-germinating treatment group;43 proteins were up-regulated and 45 proteins were down-regulated in the soil suspension treatment group.Comparing the up-regulated and down-regulated proteins of soil inhibition samples with 24-hour germination samples,It was found that 14 proteins were up-regulated at the same time,and 29 proteins were up-regulated only in the soil treatment group.17 proteins were down-regulated at the same time,and 28 down-regulated proteins in the soil treatment.In particular,five proteins were up-regulated during normal germination.Among them,three proteins?XP₀11125916.1,XP₀11125917.1,XP₀11125918.1?belong to GPI-anchored cell wall proteins?GPI-anchored cell wall proteins?,and another down-regulated protein XP₀11126608.1?GPI-anchored Phospholipase d1?,the downregulation of these GPI-anchored cell wall proteins may be one of the reasons why we tested the soil to inhibit spore germination.2 AOL_s00169g229,AOL_s00215g160,AOL_s00075g181,AOL_s00083g51,AOL_s00075g106 knockout vector were successfully constructed by electroporation of the yeast cells,Using protoplast transformation method mediated by CaC12-PEG,the positive transformants of AOL_s00169g229,AOL_s00075g181,AOL_s00083g51,AOL_s00075g106 were further confirmed by PCR.3 The mutant strains of g51s g229,g181 had no significant effect on the growth of hyphae compared with the wild type,This suggests that the KIP3,G protein alpha subunit and the aryl alcohol dehydrogenase AAD14 encoded by these genes have no significant effect on mycelial growth.The growth of mycelium is severely inhibited after knockout of the g106 gene?MAPK kinase PBS2?.The MAPK pathway is an important signal transduction pathway for all eukaryotes and plays a very important role in the growth of Arthrobotrys oligospora.4 Compared with wild type,The results showed that knockout g229 gene had no obvious effect on sporulation.g51 and g181 mutant strains,the sporulation yield was significantly lower than that of wild type strain.These two genes may be involved in the sporulation regulation of the Arthrobotrys oligospora.5 The results showed that there was no significant effect on the spore germination rate after knocking out g51 and g229 by comparing the spore germination rate of knockout and wild type.The conidia of g229 gene knockout strains were more sensitive to the inhibition of benzaldehyde and 1.3ul of benzaldehyde could inhibit the 70%spores germination,and the germination rate of wild-type spores at this concentration was 93.9%.The protein encoded by g229 was up-regulated 8.52 times,indicating that spores were able to resist the inhibitory effect of benzaldehyde by upregulating the expression level of this protein.After knocking out the g106 gene,the strains did not produce spores at all.The germinate time of g181 knockout strains was significantly delayed compared with wild type.The results showed that ammonia could inhibit the spore germination by inhibiting the expression level of these genes.The novelties of this study were described as follows:1 In this study,we analyzed the protein samples of Arthrobotrys oligospora conidia,which were extracted from ungerminated,germinated 24h,and conidial samples inhibited by soil suspension.To identify the differential proteins in each sample,and then to classify the differential proteins,functional annotations,signal pathways and metabolic pathways,and to study the molecular mechanism of soil inhibition of spore spore germination at the protein level2 By using the method of homologous recombination,the gene AOL_s00169g229,AOL_s00075g181,AOL_s00083g51,AOL_s00075g106 were successfully knocked out.And the phenotypic and physiological and biochemical characteristics of mutant and wild strains were compared and analyzed,So as to speculate the biological function of these genes.