Screening And Function Of Immune-Related MiRNAs Of Plutella Xylostella Fat Body In Response To Metarhizium Anisopliae Infection

The diamondback moth,Plutella xylostella(L.),is one of the most serious pests of cruciferous vegetables.Because of its strong reproductive ability and serious generation overlap,it is called one of the most difficult pests in the world.The misuse of pesticides has caused P.xylostella to be resistant to almost all pesticides and jeopardized the supply of green vegetables.Therefore,it is imminent to use an insecticide that is harmless to the environment and resistant to insect pests to control diamondback moth.In thisstudy,P.xylostella was used as the research material to determine the small RNA sequence36 hours after the entomogenous fungus Metarhizium anisopliae infected P.xylostella,using bioinformatics technology to screen out the differentially expressed miRNA,andaccording to immunity and development relevant target gene m RNAs were screened for the differentially expressed miRNAs of interest,and the expression profiles were verified by fluorescent quantitative PCR to construct a dynamic immune gene-miRNAs molecular network that is resistant to P.xylostella infection by M.anisopliae,laying a foundation for subsequent research.Provides a new idea for the development of fungal pesticides using the diamondback moth immune system as the target.The results are as follows:1.Obtained the optimal concentration of M.anisopliae infecting P.xylostella and the time node of sequencing.Subsequent experiments used the M.anisopliae spore suspension at a concentration of 4×10~8spores/ml to treat the 3rd instar P.xylostella for 36 hours and then the fat body was used as a sequencing template.RT-q PCR detection showed that 4×10~8spores/ml of M.anisopliae spores can significantly induce the expression of antimicrobial peptides such as Ceropin3,Defensin,Lysozyme and the immune gene Hemolin1.2.The results of small RNA sequencing showed that compared with the control,104 miRNAs were significantly differentially expressed in the fat body of P.xylostella after infection with M.anisopliae,of which 75 miRNAs were down-regulated and 29 miRNAs were up-regulated.In addition,we used RNAhybrid,miRanda,and Target Scan software to establish a standard for predicting target genes.A total of 9688 target genes were predicted.The five known miRNAs with the highest differential expression levels are:miR-1175-3p,miR-3338-5p,miR-750-3p,miR-199-x, miR-734-y.3.Annotate target genes through KEGG Orthology database and GO analysis.The signal pathways involved in the screening of three target genes may be related to immunity. Among them,miR-283-3p and miR-8545-x combine with Trypsin3(gene ID: 105397224)5’UTR region to function,and miR-8545-x It functions in the CDS region of Serine Proteinase9(Gene ID:105390003),miR-199-x binds to the CDS region of Serpin3(Gene ID:105383415),and plays an important role in the infection of insect immune fungi.4.The results of miRNA spatiotemporal expression pattern analysis show:(1)Tissue expression:miR-8545-x is highly expressed in the body wall,and miR-199-x and miR-283-3p are highly expressed in the fat body.(2)After the fungal spore suspension of M.anisopliae 10~8spores/ml infects healthy P.xylostella third instar larvae,the miR-199-x and miR-283-3p in the fat body infect the P.xylostella for 72h After that,the relative expression level reached the highest peak,while the relative expression level of miR-8545-x reached the highest value 36 hours after Metarhizium anisopliae infection.5.The analysis of the spatial and temporal expression patterns of target genes showed that:(1)Expression at developmental stage:Trypsin3 is highly expressed in male adults,followed by higher relative expression at 108h after hatching;Serpin3 has the highest relative expression during the pre-pupa stage,followed by Males;while in the larval stage,the relative expression level of the first and second instar larvae is higher than that of the third and fourth instar larvae;Serine proteinase9 has the highest relative expression level at 108h after hatching.The first and second instar larvae,and the relative expression level in the pupal stage is low.(2)Tissue expression:After detecting the target genes in the tissues of P.xylostella,it was found that the three target genes are all expressed in the body wall with the highest amount,but are almost not expressed in the midgut and Markov tube.In summary,small RNA sequencing results indicate that some miRNAs regulate the immunity of P.xylostella to M.anisopliae.through serine proteases that regulate the melanin reaction.