Molecular Mechanism Of Ecdysone 20E Titer Elevation In Bt-Resistant Plutella Xylostella(L.)

Plutella xylostella(L.)is a worldwide pest of cruciferous crops,causing an annual economic loss of 4–5 billion US dollars worldwide.Bacillus Thuringiensis(Bt)is a Grampositive bacterium,which forms a variety of protein crystals during sporulation with high efficiency and specific insecticidal activity against insects.However,due to the extensive use of Bt biologics and cultivation of Bt crops,many insects have developed resistance to them.P.xylostella is the first insect reported to develop resistance to Bt preparation in the field,Therefore,P.xylostella is a good model insect to study the molecular mechanism of insect Bt resistance.Our previous studies found that the increased content of ecdysone(20E)activated mitogen-activated protein kinase(MAPK)signaling pathway to trans-regulated the expression of midgut receptor and non-receptor genes in the Bt toxin of P.xylostella,resulting in high resistance to Bt Cry1 Ac toxin in Bt resistant strain of P.xylostella.However,the molecular mechanism of 20 E content increase remains unclear in P.xylostella.In this study,insect toxicology,molecular biology,biochemistry and molecular genetics were used to further explore the molecular mechanism of the increase of 20 E content in P.xylostella.It was found that the expression level of ecdysone oxidase gene PxEO in midgut of Bt resistant population was significantly down-regulated,and functional test confirmed that the down-regulation resulted in the increase of 20 E content,which resulted in the high Bt resistance in P.xylostella larvae.These results provide an important theoretical basis for revealing the molecular mechanism of insect Bt resistance and developing new Bt related biological products.The main research contents and conclusions are as follows:(1)Cloning and analysis of PxEO gene in P.xylostella.The gene sequence of P.xylostella ecdysone oxidase(PxEO)was obtained by Gen Bank,genome and transcriptome retrieval.The full-length sequence was obtained by PCR cloning.Stable mutations of the PxEO gene were not found by sequencing of abundant clones in the Bt susceptible and resistant strains of P.xylostella.(2)Down-regulation of PxEO gene expression was related to Bt resistance in P.xylostella.The relative expression levels of the midgut PxEO gene was significantly decreased by Quantitative Real-time PCR detection in Bt resistant strain of P.xylostella.The expression of the PxEO gene was significantly reduced by Bt Cry1 Ac prototoxin induction in the Bt susceptible strains of P.xylostella.Both RNA interference silencing of PxEO gene and 20 E treatment reduced the sensitivity of larvae to Bt Cry1 Ac protoxin in the Bt susceptible strains of P.xylostella.Genetic linkage tests showed that reduced PxEO gene expression was linked to Bt resistance in P.xylostella.(3)PxEO was correlated with 20 E in P.xylostella.The changes of the PxEO gene expression and 20 E titer showed opposite trends from the third instar larva to the pupal stage in the Bt susceptible strains of P.xylostella.In addition,RNA interference silenced PxEO gene expression,leading to the increase of 20 E content in the Bt susceptible strains of P.xylostella.And the expression of PxEO gene was significantly increased after injection of 20 E.In conclusion,the PxEO sequence of P.xylostella was identified in this study,and the decreased expression of PxEO gene was an important reason for the increase of 20 E content and Bt resistance in P.xylostella.Therefore,this study further reveals the molecular mechanism of Bt resistance in P.xylostella.