Identification Of Resistant-related Genes Of Plutella Xylostella To Bacillus Thurengensis Toxin Cry1Ac

Plutella xylostella (Linnaeus) is one of the most serious destructive and resistant development pests on cruciferous vegetables. Bacillus thuringiensis (Bt) toxins is a key insecticide which has been used to control this pest. However, P. xylostella was reported to develop rapid Bt toxin resistance, but mechanism of Bt resistance remains largely unknown, further research is required, especially Bt toxin receptor genes in resistant P. xylostella. In order to investigate functional complexity of P. xylostella transcriptome in defending Bt toxin CrylAc, an CrylAc resistance near-isogenic line ((NILs) have been established in the lab, we applied direct high-throughput paired-end RNA-sequencing to CrlAc-resistant (CrylAc-R) and-susceptible (CrylAc-S), potentially Bt-related unigenes were found in transcriptome of resistant P. xylostella, expression of these unigenes were examined by using real-time quantitative polymerase chain reactions (QPCR).1. Establishment of CrylAc resistance near-isogenic line of Plutella xylostella. Two cross methods were used to estabilshed Cryl Ac-selected resistant and isogenic susceptible strain in the lab. Method One, CrylAc resistant males and susceptible females in P. xylostella were crossed in the laboratory. The2rd instar larvae derived from the F2inbred progeny were treated with750μg/mL CrylAc toxin until it grows to pupa, the survival adults (male) after the second selection were backcrossed with the susceptible adults (female), and their inbred progeny was selected with a similar method, CrylAc resistance of the near isogenic line (NIL) were established after the process was repeated6times. Method Two, CrylAc resistant and susceptible P. xylostella were crossed6times, F6inbred progeny was separated into two strains, resistant strain were treated with dose of CrylAc toxin form low to high since2rd instar larvae every generation, another strain were treated with low dose of CrylAc toxin, susceptible adult were collected to established the isogenic susceptible strain. ISSR analysis to the4th instar larvae of the susceptible, resistant, NIL-RR, and NIL-SS strains showed the pattern of ISSR band of method One, NIL-R was similar to that of parent susceptible strain, but significantly different with the parent resistant. ISSR band of method Two, NIL-RR was similar to that of NIL-SS, but genetic variation was higher in method Two than in method One.2. Comparative transcriptional profiling of the midgut transcriptome response in CrylAc susceptible and resistant strains of Plutella xylostella. To investigate the functional complexity of the P. xylostella transcriptome in defending against the Bt toxin CrylAc, we applied direct high-throughput paired-end RNA-sequencing to two Cryl Ac-resistant (DBMlAc-R and T2-R) and one CrylAc-susceptible (DBMlAc-S) strain, the midgut transcriptome of CrylAc-resistant strain (DBMlAc-R)(GenBank accession numbers:SRX101299) was used as reference. Total of36742801clean reads was obtained in three P. xylostella strains. In resistant P. xylostella,2967for MK and2925for GK, differentially expressed unigenes (DEUs) were obtained as compared to the susceptible strain MM, among which DEUs were found significantly enriched in pathways such as metabolic pathway and biosynthesis of secondary metabolites pathway, most of the unigenes involved in the metabolic pathway exhibited up-regulated expression in resistant strains. Gene Ontology (GO) analysis revealed5892DEUs, among which105DEUs were significantly enriched with oxidoreductase activity as major molecular function category and membrane lipid metabolic process as major biological process category (P-value<0.05). In addition, Kyoto Encyclopedia of Genes and Genomes metabolic (KEGG) pathway analysis indicated that9pathways were significantly enriched in the resistant strains (Q-value<0.05). Most of the down-regulated unigenes that were potentially related to CrylAc resistance in the ABC transporter pathway were specifically located in the ABCC2and ABCC10while up-regulated unigenes were mainly associated with ABCG2. Our study will provide data to characterize and identify new genes that may be responsible for Bt resistance in P. xylostella.3. To clarify whether highly expressed, catalytic, metabolism and ABC transporters DEUs potential contribute to the CrylAc resistance of P. xylostella, the transcription levels of these unigenes in CrylAc-resistant and-isogenic susceptible P. xylostella strains were examined using real-time quantitative polymerase chain reactions (QPCR). The transcription levels of these DEUs in P. xylostella larval mid-gut were more abundant in CrylAc-resistant strain than in susceptible strain. Real-time PCR confirmed higher expression level in the resistant strain used for RNA-seq analysis, suggesting the reliability of the RNA-seq data. CrylAc resistance is potentially associated with differential expression levels of these putative Bt-related unigenes between resistant and susceptible strains of P.xylostella, CrylAc-resistant P. xylostella tends to increase catalytic, metabolic activity and ABC transporter founction in order to adapt to host plants with CrylAc toxin.