Study On The Effect Of NrMgTA Gene On Microsclerotia Formation In Nomuraea Rileyi

Nomuraea rileyi(Nr)is a double-type fungi that can infect and kill more than 40 agricultural pests of the Lepidoptera Noctuidae,and therefore has important insecticidal potential.The protein encoded by the NrMgTA gene showed that it related to physiological activities such as vacuoles,virulence and magnesium transport.However,there are few studies related to fungal MgTA genes now.In this study,the Pzp-Ptrpc-HphNrMgTA knockout vector was constructed,transformed in the wild-type Nomuraea rileyi CQNr01 by Agrobacterium,and the ΔNrMgTA mutant strain was screened.By test of theΔNrMgTA growth and development,resistance to environmental stress,pathogenicity,microsclerotia yield,etc,the specific functions of the NrMgTA gene in CQNr01 were clarified.The main findings are as follows:(1)The MgTA gene of Nomuraea rileyi closely related to that of Metarhizium.Bioinformatics analysis shows that the ORF of NrMgTA has 3288 bp,encodes 937 amino acids,encodes NrMgTA protein with a size of 106.012 KDa,the NrMgTA protein is a transmembrane protein.This gene is closely related to the homologous genes of many other species of Metarhizium.(2)The lack of NrMgTA promotes the early germination of spores and the rapidgrowth of mycelium of Nomuraea rileyi.The germination rate of the ΔNrMgTA spore in SMAY and AM was calculated,and we found that the spore germination in both mediums was faster than that of the WT,the lack of NrMgTA promoted spore germination.At the same time,the results of q PCR showed that the expression of NrMgTA in the spore of the WT was higher.The NrMgTA gene may relate to the spore germination of Nomuraea rileyi.In addition,the polar growth of the ΔNrMgTA in AM medium is much faster than that of the WT.Since AM medium provides an adverse environment,the enhanced mycelial polar growth may be a change made by the strain to adapt to this environment.(3)The lack of NrMgTA reduces the yield of MS.In the MS culture experiment,the MS yield of the ΔNrMgTA was 77.29% lower than that of the WT.The MS culture process were tracked and photographed,and it was found that the hyphae were difficult to entangle in 3-4 days.Therefore,the absence of NrMgTA gene hinders the polarity of hyphae and the formation of MS.(4)The absence of NrMgTA reduces the cell activity in the vacuole and increases the p H value in the hyphae of Nomuraea rileyi.The hyphae were photographed by transmission electron microscopy and found that no macromolecular substances and organelles to be decomposed were found in the mycelial vacuole of ΔNrMgTA,which may indicate that the lack of NrMgTA affects the substance transfer and autophagy function of the vacuole.The BCECF-AM staining experiment found that the p H value of ΔNrMgTA in the hyphae increased by about 0.822 during the microsclerotia culture process,indicating that the NrMgTA has a regulatory effect on the hyphae p H value.(4)Transcriptome differential gene expression analysis.Transcriptome sequencing analysis was performed on the products of ΔNrMgTA MS cultured to 5 d,and it was found that the expression of 5551 genes in the mutant genome had changed.Among them,the difference between genes related to iron transport and storage and mycelial polar growth is the most obvious,indicating that the absence of the NrMgTA of Nomuraea rileyi impacts the gene expression of multiple metabolic pathways.