| The diamondback moth, Plutella xylostella(Lepidoptera: Plutellidae) originated in the Mediterranean or South Africa, but is now considered a globally distributed insect pest. P. xylostella can cause crop losses of more than 90%. A recent study estimated the total annual cost of damage caused by P. xylostella to be US$4-5 billion. In China, P. xylostella has become a major pest of cruciferous crops since the 1970 s and is now found in most cruciferous crop-growing areas. Abamectin are widely used as major insectcide for the diamondback moth control since it has been found in 1970 s. However, the rapid rise in resistance to abamectin poses a serious threat to the management of the diamondback moth. A ?eld population collected in 2007 from Yunnan Province, China, exhibited about 5000-fold resistance to abamectin. The resistant strain(ABM-R) was selected continuously with abamectin. Compared to the relative susceptible strain(ABM-S), the resistance ratio was 217.68-fold.To understand the molecular basis of abamectin resistance, transcriptome analyses were carried out to evaluate the differences between abamectin resistant and susceptible strains at transcriptional level. Three biological replicates for each strains were prepared for the transcriptome sequencing. Each sample resulted in a library containing an average of ~ 76 million of reads equal to ~7.6 Gb and a total data of ~ 45 Gb were generated. A total of 219,214,576 paired-end reads(100 bp) were obtained for ABM-S, of which 109,501,256(49.95%) mapped to the P. xylostella genome. In ABM-R strain, 126,185,708(52.72%) of 239,347,474 total paired-end reads matched to the P. xylostella genome. A total of 76 and 65 up-and down-regulated transcripts, respectively, were differentially expressed(padj < 0.05) between susceptible(ABMS) and resistant(ABMR) diamondback moth. Using Blast2 GO, 141 differentially expressed transcripts were able to assign to 30 GO term. Majority of these genes were assigned to biological process, metabolic process, catalytic activity, organtic substance metabolic process and macromolecular metabolic process.Insect cytochrome P450 play an important role in catalysis of many reactions leading to insecticides resistance. Transcriptome analysis of abamectin-resistant and abamectin-susceptible in the diamondbackmoth, Plutella xylostella revealed that up-regulation of cytochrome P450 s are one of the main factors leading to the development of abamectin resistance. We report for the first time a novel cytochrome P450 gene CYP340W1, which belongs to the cytochrome P450 gene family CYP4. The full-length c DNA sequence of a P450 gene from P. xylostella was obtained and named as CYP340W1. It has a 1491 bp open reading frame(ORF), a 139 bp 5’-untranslated region(UTR), and a 299 bp 3’-UTR containing a 26 bp poly-A tail. The CYP340W1 c DNA encodes a 496-amino acid peptide with a molecular weight of 56 k Da and an isoelectric point of 9.18. CYP340W1 contain theconserved motif PFxx Gx Rx Cx G/A carries the cysteine ligand to the hemeiron, which allows the easy identi?cation of putative P450 sequences.CYP340W1 in resistance strain(ABMR) were significantly higher than that in susceptible population, which were 5.66 times higher than that in the fourth-instar larvae.The expression of CYP340W1 in P. xylostella was differentially affected by abamectin. The exposure to abamectin resulted in the largest transcript level of this cytochrome P450 gene. Expression level of CYP340W1 were increased 1.5 flod after feeding with abamectin.The findings suggested potential involvement of CYP340W1 in abamectin resistance of P. xylostella. To assess the functional role of CYP340W1 to abamectin resistance, RNA interference mediated gene silencing by double stranded RNA micro-injecting for was used. As a result of RNAi, a significant increment in mortality of larvae injected CYP340W1 ds RNA was observed after 72 h of exposure to abamectin. The bioassay of larvae injected with ds RNA indicated that the mortality of the treated 3nd instar larvae was 86.7%, which was significantly higher than that of the control group 56.7% when the abamectin concentration was 2.5 mg/L. These results strongly support that this novel cytochrome P450 gene plays an important role in abamectin detoxification in the diamondback moth and is partly responsible for its resistance.In addition, recombinant expression vector was constructed for heterogeneous expression. The Trans Setta(DE3) was used as expression hosts. The recombinant vector was transferred into hosts cell, then we optimized expression condition. The optimal conditions were determined as follows: cultivation temperature 16 ℃and concentration of 0.01 m M IPTG. Finally, Western blot confirmed that the recombinant protein was successfully expressed. |
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