Cloning And Characterization Of Ubiquitin, A Novel Deltamethrin Resistance Associated Gene From Diamondback Moth, Pluttela Xylostella (L.)

Diamondback moth (Pluttela xylostella L.), which belonging to the family Plutellidae, order Lepidopteran, has become one of the most destructive pests in Cruciferous vegetables (CFV) all over the world. Chemical control is a major method to keep insects down. However, under natural selection, continuous and excessive application of insecticides has caused the development of insecticide resistance, which is now the major obstacle to pest control. Identification and characterization of genes related to insecticide resistance will undoubtly contribute to clarify the mechanism of insecticide resistance and find the new target of insecticide resistance.In our previous experiments, we had employed cDNA representative difference analysis (cDNA RDA) to identify differentially expressed genes between deltamenthrin-resistant and-susceptible strains of Pluttela xylostella (L.), and some differentially expressed genes were isolated. One of the highly expressed genes was UBI-homolog. UBI is a small protein that ubiquitously exist in the eukaryotic cells. The primary function of UBI is to selectively degenerate proteins. UBI plays important roles in cell growth, cell cycle regulation, cell apoptosis and signal transduction. Whether UBI plays a part in insecticide resistance or what function it plays, depends on our further research.Based on these, in this study, we acquired the open reading frame of all the numbers of ubiquitin gene family by RT-PCR using Pluttela xylostella fourth instar larvae as experimental materials, we detected which gene or genes has high expression in deltamethrin-resistant strain by qPCR, and explored the relationship between ubiquitin and deltamethrin resistance (DMR) by RNA interference (RNAi) and cell transfection. The main results are as followes:(1) The RNA from diamondback moth was reversed transcribed and ubiquitin genes were cloned by RT-PCR method. The open reading frame of UBL40was387bp, which encodes128amino acids.1to76amino acids consist ubiquitin monomer, and77to128consist ribosomal protein L40. The GenBank accession number was JX163118. The open reading frame of UBS27a was468bp, which encodes155amino acids, with GenBank number was JX437934.1to76amino acids consist ubiquitin monomer, and77to155consist ribosomal protein S27a. Both UBL40and UBS27a are ubiquitin ribosome fusion proteins.(2) The results of quantitative real-time PCR revealed that UBL40is highly expressed in the deltamethrin-resistant strain of diamondback moth.(3) RNAi to resistant Pluttela xylostella fourth instar larvae was applied to silence UBL40through microinjection in vivo, and After48h, the RNAi efficiency was60%by qPCR verification.48h post RNAi, we conducted bioassay on the fourth instar lavae of resistant Pluttela xylostella. The results showed that the resistance level of RNAi group was significantly decreased compared to the control group, which means UBL40is related to deltamethrin resistance.(4) To further testify the reliability of the above results, we transfected UBL40into Drosophila Kc cells, afterwards, cells were stimulated by deltamethrin in a series of concentration gradient. Following that, cell toxicity experiment was conducted using CCK-8kit. The results showed that the survive rate of UBL40-transfected group was declined compared with controls.In sum, the results showed that UBL40is related to deltamethrin resistance, and UBL40moderate overexpression would protect cells from deltamethrin damage. Our research firstly reported that ubiquitin a pesticide resistance related gene, which provides us with a new scientific basis for clarifying molecular mechanism of insecticide resistance and establish a detection method for pesticide resistance.