Molecular Mechanisms Of PxABCBs Genes Involved In Resistance To Bacillus Thuringiensis Cry1Ac Toxin In Diamondback Moth,Plutella Xylostella (L.)

The diamondback moth,Plutella xylostella(Lepidoptera: Plutellidae),is one of the mostly pivotal pests of cruciferous crops.It can quickly develop resistance to various pesticides for high dose selection,powerful fecundity and overlapped life cycles.It is now estimated to cost the world economy US $4–5 billion annually whereas it also costs US $0.77 billion in China annually.Therefore,the effective control of the diamondback moth needs to be solved urgently for biologist.Bacillus thuringiensis(Bt)is a gram-positive bacterium which famous in biological insecticides because it can produce a variety of insecticidal crystal proteins(ICPs)that specific and high toxicity to various pests.However,unreasonably overuse of Bt often leads to Bt formulation or Bt plants.Less is known about the mechanisms of Bt resistance which delays the Integrated Pest Management(IPM)program and the development of Bt formulations.Therefore,the uncovered molecular mechanism of insect resistance to Bt has a valuable price for the IPM program and the sustainable usage of Bt products.The diamondback moth is the first insect to develop field-evolved resistance to Bt sprays and its timely publicized genome that can be acted as a good model specie for molecular mechanism of Bt resistance.According to our previous study of midgut transcriptome and RNA-seq data,the expression levels of ABC genes have significant differences between the Cry1Ac-susceptibility and the Cry1Ac-resistant strains.In our study,We firstly annotated and identified the ABCB subfamily genes from whole genome level,then functional study will be designed in significantly different expression ABCB genes in P.xylostella strains by bioinformatics,biochemistry,molecular biology molecular genetics,entomological physiology and toxicology.The results showed that reduced expression of PxABCBs genes confer high level resistance to Cry1 Ac in P.xylostella.A new insight will be provided not only for the molecular mechanism of resistance to Bt,but also provides a theoretical basis for effective pest control.The main findings are as follows:(1)Study of relationship between PxABCB1 gene and Cry1 Ac resistance in P.xylostellaIn this study,the bona fide full-length c DNA sequence of the PxABCB1 gene was cloned and analyzed,and the expression of the PxABCB1 gene was detected in all tissues and developmental stages,with the highest expression in midgut tissue and the female adult stage.Although no consistent non-synonymous mutations were identified between the susceptible and resistant strains,PxABCB1 gene expression was remarkably decreased in all resistant strains,and the association was further validated by Cry1 Ac selection in the moderately resistant SZ-R strain.Moreover,knockdown of the PxABCB1 gene expression resulted in significantly reduced larval susceptibility to Cry1 Ac toxin in the DBM1Ac-S strain,and decreased expression of the PxABCB1 gene was tightly linked to Cry1 Ac resistance in P.xylostella.(2)Characterization of PxABCB genes in P.xylostellaWith the aid of our previous transcriptome and the genome data,we analyzed and obtained 12 genes which includes eight full transporters and four half transporters.Then the phylogenetic relationship revealed that eight full transporters have a long distance with four half transporters in P.xylostella.In addition,ABCB full transporters have a complex relationship in different species while the ABCB half transporters are more conserved in evolution that one gene clustered in each other.(3)q PCR analysis and RNAi functional verification of PxABCB genes in P.xylostellaAccording to our previous midgut-transcriptome and the RNA-seq data,we obtained the expression levels of PxABCB genes in P.xylostella.Spatio-temporal expression profiles of the PxABCB genes as determined by q PCR showed that all PxABCB genes were expressed differently.Then the PxABCB9 gene expression was remarkably increased(6-fold)while the other genes,PxABCB4,PxABCB8 and PxABCB11,were significantly reduced in all resistant strains.Moreover,knockdown of the PxABCB4 gene expression resulted in significantly reduced larval susceptibility(almost 8%)to Cry1 Ac toxin in the DBM1Ac-S strain,but knockdown of the PxABCB8 gene have no significant difference.All in all,the above results clarifies the roles of PxABCB genes,which acted as a receptor,to Bt Cry1 Ac resistance in P.xylostella and provide a new vision for the molecular mechanism of Bt Cry1 Ac in insects.