The Knockout And Analysis The Function Of G Protein Coupled Receptors In Metarhizium Robberstii

Metarhizium is playing an important role in biological control of pests of an important class of insect pathogenic fungi,not only have all the advantages of fungi,but also have the advantages of safety,easy production,long shelf life and so on.So it was widely studied.According to incomplete statistics,more than 200 kinds of agricultural and forestry pests in the world can be killed by the Metarhizium.However,Metarhizium with other micro-organisms as pesticides,there are also some shortcomings.Compared with chemical pesticides,insects,fungal pathogens developed lasting insecticide action time,no environmental pollution,safety of non-target organisms,but there are also a serious impediment to the fungus the following shortcomings of the wide application of pesticides:death for a long time to kill insect low efficiency,control effect of instability,to address this issue,reveal the insects at the molecular level is particularly important pathogenic mechanism of pathogenic fungi.,the efficiency is low,the effect is not stable,and so on.Therefore,the mechanism of the insect pathogenic fungus can be further understood,which can provide a theoretical basis for the breeding of highly effective insecticidal strains.G protein coupled receptors(GPCRs)is a kind of very important signal molecule receptor.It is the biggest kind of transmembrane protein family in eukaryotes.G protein coupled receptors play an important role in the transduction process of extracellular signal to the cell and regulating the transcription,cell movement and growth of cells.It can be said that GPCRs regulates almost all known physiological processes,such as hormones,growth factors,neurotransmitters,smell and light,and so on.Therefore,G protein coupled receptors have an important role to play in vivo.Although filamentous fungus is the lowest part of eukaryotes,it has a profound influence on the human life activities.Therefore,it is necessary to further understand the physiological,genetic and molecular biology of filamentous fungi,and to carry out effective reform in order to control the harmful effects to the human beings of filamentous fungi.In recent years,the whole genome sequencing of a lot of filamentous fungi has been done or is being done,so the research on specific gene function has become a hot spot in the research of filamentous fungi.Wild type spore suspension of Metarhizium robberstii ARSEF 23(Mr23)in 40 ℃water bath heat shock,in the analysis of the expression profiles,the formation of after heat shock,results showed that the expression of G protein coupled receptor gene is up-regulated,so we can speculate that in the process of antiadversity of Metarhizium,the G protein coupled receptor plays a very important role.In this paper,the gene function of G protein coupled receptors in wild-type Metarhizium robberstii ARSEF 23(Mr23)was predicted by the combination of gene knockout and Agrobacterium mediated genetic transformation.First,we find the location of GPCRs in the genome,and find its upstream and downstream sequences,and construct the knockout vector,which can be transformed into Agrobacterium(AGL-1),and obtain the Agrobacterium strain,which contains knockout plasmid.The Agrobacterium strain and the Metarhizium robberstii ARSEF 23(Mr23)were co-culture.The positive strains were selected,which is knockout strains,and we used the same method to construct a anaplerosis mutation Agrobacterium strain,which was co-culture with knockout strains of Metarhizium.Then we obtain the revertant mutant strain of Metarhizium.The wild type strains of Metarhizium robberstii ARSEF 23(Mr23),knockout strains(△MrGPCR)and revertant mutant strain(△MrGPCR::MrGPCR)were determined by the corresponding biological properties,determination,determination of anti inverse performance and ability to observe and compare the corresponding virulence,arrival at a conclusion.From the present situation,the application of GPCRs in the fungus is still limited,so it is very promising and valuable to study the GPCRs in the Metarhizium.So the research on the function of GPCRs in the Metarhizium has a certain theoretical significance and practical application value.The main results are as follows:1.The sequence of G protein coupled receptors was found in NCBI,the location of protein was found in the genome.And constructing the knockout plasmid and the anaplerosis plasmid,obtained the knockout strain and the revertant mutant strain.2.Wild type strains of Metarhizium robberstii ARSEF 23(Mr23),knockout strains(△MrGPCR)and revertant mutant strain(△MrGPCR::MrGPCR)were selected for 14 days of the strain,and configured they to spore suspension,using 30μL spore suspension inoculation on the SDAY,1/4 SDAY and PDA medium(including 3 groups biological repeat and 3 groups of technology repeat),to calculat the sporulation at 14 days and 20 days respectively.The results showed that compared with the wild type Metarhizium(Metarhizium robberstii ARSEF 23),sporulation of knockout of GPCR(△ MrGPCR)decreased significantly.3.Wild type strains of Metarhizium robberstii ARSEF 23(Mr23),knockout strains(△MrGPCR)and revertant mutant strain(△Mr GPCR::MrGPCR)were inoculated on SDAY medium,were extracted the RNA from them,to find relevant sporulation genes by real-time fluorescence quantitative PCR method,the results showed.The most relevant and sporulation genes have a downward trend,proving above-mentioned result(conclusion 2)by molecule that the amount of spore production decreased significantly.4.The spore suspension of wild type strains of Metarhizium robberstii ARSEF 23(Mr23),knockout strains(△ MrGPCR)and revertant mutant strain(△ Mr GPCR::MrGPCR)point contact on the PDA medium what obtain some kinds of reagent.Correspond every other time to observe the growth of the colony.The results showed that on the medium of NaCl,SDS,Congo red and H2O2 and carbendazim resistance,compared with the wild type Metarhizium(Metarhizium robberstii ARSEF 23).Adaptability of knocked out of GPCR(△MrGPCR)to the surrounding environment has become stronger.The sensitivity of adversity has declined.5.The spore suspension of wild type strains of Metarhizium robberstii ARSEF 23(Mr23),knockout strains(△ MrGPCR)and revertant mutant strain(△ Mr GPCR::MrGPCR)spread on the PDA medium uniformity.After 14 days of culture,collecting spores,configured they to spore suspension,with fresh spore suspension to immersion in wax moth,wax moth infection on body surface.The results showed that,compared with the wild type Metarhizium(Metarhizium robberstii ARSEF 23),infection of Galleria mellonella capacity of knocked out of GPCR(△ MrGPCR)has decreased,which reduced virulence.In summary,knocked out of Metarhizium GPCR(△Mr GPCR)adaptive variation in adversity,reduced the amount of spore production,attenuated virulence.