Primary Research On Regulatory Pathway Of Microcycle Conidiation In Metarhizium Acridum

As an entomopathogenic fungus,Metarhizium acridum infects locusts by conidia.Therefore,the research of its conidiation mechanism plays an important role in the development and application of biological pesticides.Filamentous fungi include two conidiation methods: normal conidiation and microcycle conidiation.Normal conidiation is the most common conidiation pattern of filamentous fungi,and its conidiation mechanism has been clarified.Microcycle conidiation is a kind of asexual sporulation method that the fungus bypasses normal life history.Metarhizium acridum showes microcycle conidiation in different inducing conditions,and it has the advantages of rapid conidiation rate,large sporulation,and uniform conidia size compared with normal conidiation.In recent years,the influence of environmental factors and related genes on microcycle conidiation has been studied,but the regulatory relationships between related genes and the regulatory mechanisms have not been reported.1.Research on Regulatory Relationship between Genes Related to Microcycle Conidiation in Metarhizium acridumIn this laboratory,the microcycle conidiation medium,SYA,was selected in the previous period.A homogenized full-length c DNA library and an enriched library of genes related to microcycle conidiation were constructed and identified.The genes,Ma H1,Ma Cre A,Ma AGA,Ma PPT5,Ma Cpp,Ma Mmc,Ma Rcp,were involved in the shift between normal conidiation and microcycle conidiation in M.acridum,and the disruption strains were screened.In this study,we observed conidiation differences between the disruption strains and wild-type strains on 1/4SDAY and SYA respectively,and the relationship between genes was analyzed by q RT-PCR.The main results are as follows:(1)There are differences in conidiation patterns between ΔMa H1,ΔMa PPT5,ΔMa Rcp and WT strains on SYA medium.WT strains exhibit normal conidiation,while ΔMa H1,ΔMa PPT5,ΔMa Rcp strains present microcycle conidiation.(2)There are differences in conidiation patterns between ΔMa Cre A,ΔMa Cpp,ΔMa Mmc,ΔMa AGA and WT on SYA medium.WT strains exhibit microcycle conidiation,while ΔMa Cpp,ΔMa Mmc,ΔMa AGA strains show normal conidiation.Especially,ΔMa Cre A presents mycelial growth without conidial.(3)Conidia or hyphae of ΔMa H1,ΔMa PPT5,ΔMa Rcp and WT strains on 1/4 SDAY medium were collected and total RNA was extracted.The results by q RT-PCR analysis showed that Ma Rcp and Ma PPT5 genes regulate each other,while Ma H1 has no relationship between them.(4)Conidia or hyphae of Ma Cre A,Ma Cpp,Ma Mmc,Ma AGA and WT strains on SYA medium were collected and RNA was extracted.The results were obtained by q RT-PCR,which Ma Cre A is a central regulatory gene for microcycle conidiation that regulates the expression of Ma Cpp,Ma Mmc,and Ma AGA.Meanwhile,Ma AGA is a key gene for the regulation of microcycle conidiation,and its expression is regulated by Ma Cre A,Ma Cpp,and Ma Mmc.2.Functional analysis of Ma AGA and its molecular mechanisms for regulation of microcycle conidiationArginase hydrolyzes arginine to ornithine and urea,while arginine is the only endogenous nitrogen-containing substrate of NOS.In microorganisms,such as fungi that lack urea cycle,arginase regulates the use of nitrogen sources and conidiation processes.However,the regulation mechanism of arginase on microcycle conidiation in filamentous fungus has not been reported.ΔMa AGA strain were constructed in previous study,and the influence of Ma AGA on microcycle conidiation was found.In this study,Ma AGA complement strains were obtained.The function of Ma AGA and the molecular mechanism of regulating microcycle conidiation were further studied by the analysis of growth,virulence,stress,NOS activity and NO content.The main results are as follows:(1)It was found that the early germination rate of ΔMa AGA was higher than that of WT on SYA medium through the analysis of growth characteristics,but Ma AGA did not influence the germination on 1/4SDAY media.Through virulence analysis,it was found that Ma AGA does not affect the virulence of M.acridum.The analysis of stress resistance showed that Ma AGA did not affect the UV resistance of M.acridum,but the wet-heat tolerance of ΔMa AGA was significantly higher than that of WT and CP strains.(2)Constructing Pgpd A-Ma AGA-EGFP fusion expression vector,and the green fluorescence observation showed that Ma AGA was localized in the cytoplasm.(3)The colony growth and conidiation type of ΔMa AGA did not return to microcycle conidiation on SYA medium exogenously added ornithine and urea.(4)The NOS activity and NO content of ΔMa AGA were significantly lower than that of WT when ΔMa AGA strain showed normal conidiation.(5)The conidiation type of WT converted to normal conidiation from microcycle conidiation on SYA medium exogenously added with L-NNA.Meanwhile,the conidiation type of ΔMa AGA converted to microcycle conidiation from normal conidiation on SYA medium exogenously added with SNP.All this results demonstrated that the disruption of Ma AGA reduced NO content and then regulated microcycle conidiation in Metarhizium acridum.