| Plutella xylostella(L.)belongs to Lepidoptera,which is a cosmopolitan agricultural pest.Bacillus thuringiensis(Bt)is one of the most widely used insecticide,which made remarkable achievements in the control of P.xylostella.However,with the large-scale application of Bt agents and widely plant of Bt transgenic crops,the resistance to Bt in insects is seriously increased.P.xylostella is the first species with Bt resistance in the field.Insuficient understanding of the resistance mechanism of Bt restricts the further exploitation and application of novel insecticidal proteins,moreover,it also seriously threatens the utilization of Bt transgenic crops.In order to study the mechanisms of Bt toxin resistance,the near-isogenic P.xylostella with>4,000-fold resistance were used for whole-genome methylation sequencing,transcriptome sequencing and miRNA sequencing,the main results are as follows:1)We estimate that the whole-genome methylation level is 0.8%,the majority methylated sites occur in CG dinucleotides,and the methylation level in resistant strain is lower than Cry1Ac susceptible strain.mCG mainly concentrated in the gene regions(especially in exon regions),and the majority of mCG have low levels of methylation.CpG Methylation is enriched in the 5 ’ coding region of methylated genes.425 differentially methylated genes(DMGs)between Cry1Ac resistant and susceptible strains were identified,about 65.4%DMGs in resistant strain are hypomethylation.In these hypomethylation genes,six candidate insecticide resistance genes were identified.2)437 differentially expressed genes between Cry1Ac resistant and susceptible strains were obtained,the functions of these genes are conserved,mainly enriched in oxidoreductase activity,protein degradation activity and peptidase activity.A lot of potential Bt receptor and candidate insecticide resistance proteins in the differentially expressed genes were discovered,include ATP-binding cassette(ABC)transporter,aminopeptidase N(APN),alkaline phosphatase(ALP),MAPK-related genes,peptidase,trypsin,chymotrypsin and so on.We investigated the relationship between the methylation and gene expression and found that gene expression is correlated with methylation status,but independent of methylation level.The expression in methylated genes were significantly higher than non-methylated genes,suggest that CpG methylation are critical for the expression of genes.3)We identified 153 novel miRNAs,of which 34 are conserved between P.xylostella and other 25 insects.A total of 13 novel and differential expressed miRNAs were identified between Cry 1Ac resistant and susceptible strains.Target prediction analysis revealed that these miRNAs participate in binding and catalytic activity,which is consistent with the results of proteome analysis,indicating that miRNA may play an important role in post-transcriptional regulation.We also compared the methylation level between target and non-target genes of differential expression miRNAs,and found that the methylation level of target genes were significantly higher than non-target genes,indicate that miRNA may play a role in gene methylation. |
