Transcrptional Regulation Mechanisms Of Midgut Bt Cry1Ac Toxin Receptor Genes In Plutella Xylostella (L.)

The insecticidal crystal proteins generated by the gram-positive soil bacterium Bacillus thuringiensis(Bt)can specifically kill various insect pests without causing harm to non-target organisms or the environment;as such,Bt biopesticides and genetically modified Bt crops have been widely used for pest control.Unfortunately,different insect pests in the field have developed high-level resistance to Bt sprays or Bt crops,gravely threatening the sustainable application of Bt products.The diamondback moth Plutella xylostella(L.)is a global pest of cruciferous crop and is the first documented insect developing field-evolved resistance to Bt biopesticide.Recent studies have shown that the down-regulation of midgut Cry receptor genes including PxABCB1 and PxABCG1 reduces toxin-receptor interaction and thus leads to Bt resistance in the diamondback moth.However,little is known about the mechanisms of transcriptional regulation of these midgut Cry receptor genes.Here,we explored the mechanisms of transcriptional regulation of the differential expression of the midgut Cry receptor genes PxABCB1 and PxABCG1 in P.xylostella,revealing that the transcription factor PxJun activated by the MAPK signaling pathway increased the resistance of P.xylostella to Cry1 Ac by inhibiting PxABCB1 expression,and that a cis-acting mutation in the PxABCG1 promoter was associated with Cry1 Ac resistance in P.xylostella.These studies contribute to a better understanding of the transcriptional regulation of midgut Bt toxin receptors and the molecular mechanism of insect Bt resistance,which is essential for the sustainable application of Bt biotechnology and the integrated pest control.The main findings are as follows:(1)Cloning and activity analysis of the PxABCB1 promoter in P.xylostellaBased on the genomic data of P.xylostella,we cloned the promoter sequences of the PxABCB1 gene in Cry1 Ac susceptible strain DBM1Ac-S and resistant strain NILR.The promoter activity was detected by a dual-luciferase reporter assay,and showed that there was no significant difference in promoter activity between susceptible and resistant strains.Further finely truncated promoter recombinant plasmids were constructed to identify the essential regulatory region of the PxABCB1 gene.(2)The transcription factor PxJun negatively regulates PxABCB1 promoter activityA series of potential binding sites for transcription factors were predicted in the important regulatory regions of PxABCB1 promoter using online software.To study the effects of these transcription factors on the promoter activity of the PxABCB1 gene,we cloned the coding regions of these genes,and then constructed their corresponding expression vectors and carried out a dual-luciferase reporter assay.The result showed that PxJun negative regulated PxABCB1 promoter activity.The mutation or deletion of the predicted Jun binding site(JBS)caused PxJun to be unable to inhibit PxABCB1 promoter activity.The yeast one-hybrid assay(Y1H)showed the direct interaction between PxJun and the JBS.(3)MAPK-activated PxJun represses PxABCB1 expression and enhances the larval resistance to Cry1 Ac toxinReal-time quantitative PCR(qPCR)revealed that PxJun was expressed in different tissues and developmental stages in P.xylostella.Furthermore,the midgut PxJun expression in all Bt resistant strains was higher than that in the susceptible strain.Silencing of the PxJun gene in the Bt resistant strain NIL-R significantly increased PxABCB1 expression and enhanced larval susceptibility to Cry1 Ac,while PxMAP4K4 silencing significantly reduced PxJun expression and increased PxABCB1 expression.(4)Cloning and activity analysis of the PxABCG1 promoter in P.xylostellaBased on the genomic data of P.xylostella,we cloned the promoter sequences of the PxABCG1 gene,and obtained the main-type promoters in the susceptible strain(S1and S2)and the resistant strain(R2).The dual-luciferase reporter assay was conducted and showed that the promoter activity of S1 and S2 was significantly higher than that of R2.Further study found that the key region leading to differential activity was between-972 and-891.Sequence alignment analysis showed that there was a single nucleotide polymorphism(SNP)in this region.Further predictions of transcription factor binding sites found that there was an Antp/Dfd binding site in the susceptible strain,whilst the cis-mutation in this element led to the disappearance of this motif in the resistant strain.(5)The transcription factor Antp is involved in the regulation of the PxABCG1geneTo investigate whether Antp and/or Dfd regulate the PxABCG1 gene,we amplified the coding region sequences of Antp and Dfd and constructed the corresponding expression vector to perform dual-luciferase reporter assays.The results showed that Antp significantly enhanced the S1 activity,but had little effect on R2.Antp could not trigger the activity of the PxABCG1 promoter S1 after deletion or mutation of the predicted Antp binding site(CRE).The Y1 H further verified the interaction of Antp with normal CRE but not mutant CRE.After Antp silencing by RNAi,the expression of the PxABCG1 gene was decreased significantly in the susceptible strain;however,down-regulation of Antp did not have an effect on PxABCG1 expression in the resistant strain.These results indicate that a cis-mutation in the PxABCG1 promoter eliminates the binding and regulation of Antp,thereby decreasing the expression of the PxABCG1 gene.