Study On The Role Of Plutella Xylostella MiRNA In The Immunity Against Paecilomyces Cicadae Infection

The diamondback moth(DBM),Plutella xylostella(L.)(Lepidoptera: Plutellidae),is a major pest of cruciferous vegetables and is known to cause serious losses in agricultural production.Due to the long-term use of chemical control coupled with the intensive and irrational use of insecticides,P.xylostella has developed resistance to various types of insecticides and has become one of the most resistant pests in the world.Therefore,the development of biological control technology has become an inevitable trend to control P.xylostella.Insects mainly rely on the innate immune system to defend against the invasion of pathogenic microorganisms.If the insect loses its innate immunity,its survival will be affected.Entomopathogenic fungi and its virulence factors are used for controling pests.Analyzing host insects' immune defense mechanism against entomopathogenic fungi can provide theoretical basis and possible target for entomopathogenic fungi to control pests.Micro RNAs(miRNAs),a group of non-coding RNAs,19-24 nt in length,modulate many biological processes,including development,metabolism,behavior,metamorphosis and immunity,are regulated by post-transcriptional regulation to degrade m RNA or block translation.In this study,P.xylostella was selected as the research object.The small RNA of P.xylostella induced by Paecilomyces cicadae was sequenced,and the miRNA was identified.The differentially expressed miRNAs were analyzed,and immune-related target genes of miRNAs were predicted.The expressions of Toll pathway immune genes after microorganisms induction were analyzed.The RNAi method was used to investigate the function of pxy-miR-8523?1ss16TG on xSpz5 gene expression regulation.The main results were summarized as follows:1.miRNAs of P.xylostella induced by P.cicadae induced were sequenced.432 miRNAs were identified,among which 248 were known miRNAs and 184 miRNAs were newly discovered.There were 32 significantly differentially expressed(p <0.05)miRNAs.The results of RT-q PCR showed that the expression levels of bmo-miR-13b-3p and PC-5p-282993?8 were significantly higher than the control under the induction of bacteria and fungi.The expression levels of three miRNAs of bmo-miR-14-3p?R+1,pxy-miR-8523?1ss16TG and bmo-miR-1a-3p under the induction of different microorganisms were significantly lower than those of the control group.2.The predicted miRNA immune-related target genes included Toll pathway,IMD pathway,JNK pathway,JAK-STAT pathway,pattern recognition protein and antibacterialeffect factor.Toll pathway genes included myd88,Toll-6,Toll-7,spz3,spz4,spz5,pll / tube,a PKC,ref(2)P,ECSIT,and UBA2.The target gene of pxy-miR-8523?1ss16TG was xSpz5.3.Molecular characteristics of Toll pathway-related genes:(1)xSpz5 c DNA sequence ORF was 1281 bp long,encoding 426 aa,mainly expressed in fat body and midgut.xSpz5 was highly expressed in third and fourth instars.P.cicadae and Staphylococcus aureus can induce up-regulation of xSpz5 expression.(2)Toll pathway immune genes such as Px Toll,Px Tube,Px Dorsal and Px Cactus were up-regulated under the induction of bacteria and fungi.4.Results of RNAi of xSpz5 showed that the transcription level of xSpz5 gene decreased after ds xSpz5 injection compared with ds GFP and DEPC water treatment.Compared with the control group,the death rate of the ds xSpz5 treatment group was significantly increased.The survival rate was 63.3% post infection 48 h,which was significantly lower than the control group,DEPC water group,and ds GFP group.5.The mimic and inhibitor of pxy-miR-8523?1ss16TG was injected into P.xylostella,respectively.RT-q PCR rsults showed that the expression level of xSpz5 decreased in the injection group of pxy-miR-8523?1ss16TG mimic,while the expression level of xSpz5 was up-regulated in the injection pxy-miR-8523?1ss16TG inhibitor group.Compared with the control group(mimic-NC injection),the mimic injection group infected by P.cicadae showed asignificantly increased mortality.After 48 hours of infection,the survival rate of larvae of the pxy-miR-8523?1ss16TG mimic group was 61.7%,which was significantly lower than that in the mimic-NC and CK groups.Through this study,the analysis of the function of host insect miRNA in the immune mechanism can provide possible targets and some theoretical basis for the use of insect pathogenic fungi and mycotoxins to control pests.It will also provide ideas for new pest biological control methods.