Study On The Clone,prokaryotic Expression Of Plutella Xylostella's PxEH Gene And The Cry2Ab's Insecticidal Activities By RNA Interference

Pathogens activate the immune system of insects during infection.Cry toxin is a kind of insecticidal protein that can form channels in the midgut of target insects.At present,the mechanism of insect immune response to Cry toxin is still unclear.In our study,a key gene(Plutella xylostella poly(U)-specific endoribonuclease homolog)involved in immune response is cloned from the midgut DNA of Plutella xylostella.The expression of the immune gene increases when Plutella xylostella is infected with the pathogen,which protects Plutella xylostella by immune response.Sequence analysis shows that the open reading frame of the gene is 1053 bp in length and encodes 350 amino acids.We name it PxEH.The homology between Plutella xylostella poly(U)-specific endoribonuclease(XM 011563907.1)on NCBI and PxEH is 97%.We use the cloned PxEH immune gene to construct Prokaryotic expression vector pGEX-4T-1/PxEH.The target protein is expressed in B121 expressing bacterna.The molecule weight of the fusion protein is 66 kDa,and the expression level is relatively high.It is confirmed by Western blot experiment as the target protein with GST tag.The conditions to express PxEH immunogenic protein are optimized.The most amount of PxEH immunogenic protein is expressed at 25 ?,0.2 mmol/L IPTG.The immune protein PxEH with GST tag is purified by GST agarose column.This is a new type of immune protein expressed in prokaryotic expression of Plutella xylostella gene.In order to study the role of this gene in Cry toxin insecticidal process,we silence the expression of PxEH gene by RNAi.Our study first selects a fragment of Plutella xylostella elongation factor EFla as an internal reference gene.The expression of homologous immune genes of Plutella xylostella is confirmed by qPCR that its expression will change with Plutella xylostella's uptake of Cry protein.The PxEH gene of Plutella xylostella increases to more than 8 times 1 h after intake of Cry protein,and then still increases with time but relatively insignificant.It is speculated that PxEH gene,as an immune gene,participates in the process of the immune stress response to protect Phitella xylostella itself when it uptakes Cry toxin.As time goes by,the up-regulation amount of immune genes of Plutella xylostella decreasesDsRNA is synthesized by mega scrip RNAi kit in vitro.Our study optimizes the amount of synthesized dsRNA and the optimal time for the down-regulation of expression of dsRNA in Phitella xylostella.The results show that only 0.5-fold dsRNA could silence PxEH gene's expression of Phitella xylostella.It is speculated that there is a PC R-like amplification step when dsRNA interferes with Phitella xylostella.The expression of PxEH decreases significantly to below 0.4 within 3 hours of feeding dsRNA.The best interference time is within 3 hours.With time going,the degradation of dsRNA easily leads to no interference.The insecticidal activity of feeding Cry2Ab protein and dsRNA is tested.The mortality rate is counted 48 hours later.The results show that the insecticidal activity of Cry2Ab protein to Phitella xylostella increases after RNAi silences the expression of PxEH.The LC50 decreases from 0.0103 ?g/ml to 0.0089 ?g/ml.After RNAi,the sensitivity to Cry2Ab protein increases and the mortality increases.The sensitivity of Phitella xylostella to Cry2Ab insecticidal protein increases after the expression of PxEH is silenced,which is also consistent with expected results.This study provides an experimental basis for RNAi silencing PxEH gene of Phitella xylostella to enhance the insecticidal toxicity of Cry toxin to Phitella xylostella.In our study,immunofluorescence technique is used to study where the Cry protein in the midgut of Plutella xylostella is binding.The result shows that Cry protein binds to the internal and external membrane of the midgut of Phitella xylostella,and BBMV is presumed to exist on internal and external membrane.