The Molecular Mechanism Of MiR-2b-3p Targeting Trypsin-9 To Regulate The Toxin Activation Of Cry1ac In Plutella Xylostella.L

Plutella xylostella(Plutella xylostella L.)is a worldwide pest of cruciferous vegetables,which causes serious economic losses to global vegetable production.In recent years,due to the unreasonable use of chemical agents,the moth has developed resistance to a variety of pesticides.Bacillus thuringiensis(Bt)is a gram-positive pathogenic microorganism,and the insecticidal crystalline protein produced by Btduring sporulation can specifically kill a variety of pests.The diamondback moth was the first pest to develop resistance to Btpesticide in the filed.Currently,in the reports of diamondback moth resistance to Bt,the crystal proteins involved are mainly in Cry.In particular,the research on the mechanism of Cry1 Ac resistance is comprehensive,but mainly focuses on the changes in the binding of Btactive toxin to the cell membrane receptor of the midgut epithelium,while the research on the changes in the activation of the original toxin and the Cry toxin of the midgut immunity of pests is less.Trypsin is an important proteolytic enzyme in most species,which plays an important role in protein digestion and immune defense,and also participates in the activation and toxicity regulation of Btoriginal toxin.micro RNAs(miRNAs)are widely involved in the regulation of gene expression in all eukaryotes through the degradation and translation inhibition of target gene mrnas at the transcriptional level.However,studies on miRNA regulation of Trypsin gene expression to activate Cry protoxin have not been reported.In this paper,the molecular mechanism of miR-2b-3p targeting PxTrypsin-9 to regulate the midgut immune Cry1 Ac of Plutella xylostella was studied by means of molecular biology and bioinformatics.The results show that PxTrypsin-9 plays an important role in the activation and digestion of Cry1 Ac prototoxin.The main results are as follows:1.Analysis of the RNA-seq data of the transcriptome of the middle intestine of the small vegetables moth after Cry1 Ac treatment in this laboratory revealed that the expression of trypsin-9 gene(named as PxTrypsin-9)was significantly down-regulated and negatively correlated with the significantly differentially expressed miR-2b-3p.miRanda,RNAhybrid and Target Scan predicted that miR-2b-3p targeted PxTrypsin-9,and the target sequence was consistent.This suggests that miR-2b-3p negatively regulates its expression by targeting con-sistent.This suggests that miR-2b-3p negatively regulates its expression by targeting PxTrypsin-9.Subsequently,we cloned the gene and analyzed the homology of its amino acid sequence.It was found that Pxtrypsin-9 and On Try9 had a high homology,suggesting that the protein functions were close.2.The expression pattern of PTxrypsin-9 gene was detected by qRT-PCR.?:PxTrypsin-9genes are expressed in DBM im high,other organizations express quantity is low;The expression of PxTrypsin-9 gene was the highest in the larval stage,but low in other developmental stages.The highest expression of PxTrypsin-9 gene was found in the fourth instar(gluttony phase)of larvae at different developmental stages.The results showed that Pxtrypsin-9 gene was involved in the food digestion of the diamond-moth.??:Cry1Ac original toxin infect DBM PxTrypsin-9 gene expression can be reduced significantly.However,the expression of miR-2b-3p was significantly increased,indicating that the expression of Pxtrypsin-9 gene was negatively correlated with the expression of miR-2b-3p after Cry1 Ac infection.3.Further studies on the construction of a dual luciferase reporter system in vitro confirmed that in the presence of miR-2b-3p mimics,the transcription level of PxTrypsin-9significantly decreased.miR-2b-3p gene expression was inhibited and PxTrypsin-9 gene expression was up-regulated at 24,48 and 60 h of addition inhibitor.4.Further studies on the function of PxTrypsin-9 gene by RNA interference(RNAi)technology were conducted.The synthesized ds RNA-PxTrypsin-9 was injected into the larva of diamond-moth by microinjection,and the expression of px Trypsin-9 gene was significantly down-regulated after 24 h.At the same time,ds RNA24 injection was used to incubate the midgut solution with the original toxin Cry1 Ac for 1h,and sds-page results showed that the activation and digestion ability of the midgut solution to the original toxin Cry1 Ac was decreased.The results showed that pxtrypsin-9 plays an important role in the activation of Cry1 Ac and the prevention of bacillus thuringiensis infection in the midgut of the moth.5.This paper constructs the p ET-32a-PxTrypsin-9 recombinant prokaryotic expression vector,and through the host bacterium BL21 expression under the condition of IPTG induction,SDS-PAGE detection PxTrypsin-9 protein is expressed in large quantities under IPTG induction,molecular weight of 45 k Da,with resin with 6×His label for the purification of recombinant proteins,for later PxTrypsin-9 laid a solid foundation for the further study of the protein function.In this paper,the targeted regulation of miR-2b-3p on px Trypsin-9 gene and the correlation between px Trypsin-9 and Btprimordium toxin activation were clarified,indicating that px Trypsin-9 plays an important role in the defense mechanism of the cabbage moth against bacillus thuringiensis infection.The results provided a theoretical basis for further elucidating the BtCry resistance generation and the involvement of miRNA targeting specific genes in pest and disease control.