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Cloning and Characterization of Ubiquitin Ribosome Fusion Gene Rps27a, a Deltamethrin- Resistance–Associated Gene from Diamondback Moth (Plutella Xylostella L.)

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Cloning and Characterization of Ubiquitin Ribosome Fusion Gene Rps27a, a Deltamethrin- Resistance–Associated Gene from Diamondback Moth (Plutella Xylostella L.) Turkish Journal of Zoology Turk J Zool (2013) 37: 506-513 http://journals.tubitak.gov.tr/zoology/ © TÜBİTAK Research Article doi:10.3906/zoo-1209-2 Cloning and characterization of ubiquitin ribosome fusion gene RpS27a, a deltamethrin- resistance–associated gene from diamondback moth (Plutella xylostella L.) 1 1 2 1 1 1, Hong ZHANG , Chen CHENG , Fengliang LI , Shuyu GU , Zhou ZHOU , Luogen CHENG * 1 Jiangsu Key Laboratory of Biodiversity and Biotechnology, College of Life Science, Nanjing Normal University, Nanjing, P.R. China 2 Institute of Plant Protection, Guizhou Academy of Agricultural Sciences, Guiyang, P.R. China Received: 04.09.2012 Accepted: 07.04.2013 Published Online: 24.06.2013 Printed: 24.07.2013 Abstract: The ubiquitin ribosome fusion gene RpS27a’s coding sequence was cloned from diamondback moth, Plutella xylostella (L.). An open reading frame of 468 bp was found to encode a putative 155 amino acid precursor protein, which shares over 90% similarity with other insects’ RpS27a protein. Real-time quantitative PCR was carried out to determine the relative expression level in the fourth instar larvae of deltamethrin-resistant and deltamethrin-susceptible strains. The results showed that the expression of RpS27a was significantly higher in the deltamethrin-resistant strain than in the deltamethrin-susceptible strain. There are reports indicating that upregulation expression of some ribosomal proteins confers some insecticide resistance. For the first time, we have predicted that the increased expression of the RpS27a gene may have some association with pesticide resistance in Plutella xylostella. Key words: Plutella xylostella, ubiquitin ribosome fusion gene, insecticide resistance, cDNA cloning, real-time quantitative PCR 1. Introduction responsible for resistance to these compounds. This type In recent years, the diamondback moth, Plutella xylostella of resistance is termed knockdown resistance and was first (L.), has been the most destructive pest of Cruciferae crops observed in Musca domestica (Busvine, 1951), then later all over the world; the annual cost from it is prodigious found in P. xylostella (Hama et al., 1987; Schuler et al., 1990). (Talekar and Shelton, 1993). Chemical control is now the Many studies have indicated that metabolic resistance is main method for managing insect pests in agriculture. closely related to enhanced activity of P450 enzymes (Shen The synthetic pyrethroid-class pesticides, including et al., 2003), nonspecific esterases (Hemingway et al., 2000; deltamethrin, are the most effective insecticides and are Paton et al., 2000), and glutathione S-transferases (Vontas commonly used in agriculture as well as in the household, et al., 2001). In addition, more genes may be involved because they have demonstrated limited pesticide in insecticide resistance, and the candidate genes must persistence in soil, high efficiency in killing pests, and low be identified. To investigate insecticide resistance in the toxicity to humans and other mammals (Soderlund et al., diamondback moth, we previously have separated several 2002; Sayeed et al., 2003; Yang et al., 2008). Unfortunately, genes using cDNA representational difference analysis resistance to insecticides has appeared after their long- (Cheng et al., 2005). One of the differentially expressed term use and massive application, which is now a major genes in Plutella xylostella is of great homology with the obstruction and problem in pest control (Widawsky et al., ubiquitin gene. 1998; Moulton et al., 2000). Ubiquitins, whose fundamental role is in mediating Resistance to pesticides is a polygenic inheritance intracellular selective protein degradation by the ubiquitin phenomenon. Traditionally, types of resistance have proteasome system, are highly conserved in all eukaryotes been mainly divided into 3 classes: reduced cuticular investigated (Ozkaynak et al., 1987; Binet et al., 1991). penetration (Noppun et al., 1989), target site resistance They are encoded by 2 classes of genes: polyubiquitin (mutations in the target genes) (Soderlund and Knipple, and ubiquitin ribosome precursor protein genes, whose 2003), and metabolic resistance. The voltage-sensitive primary translation products are fusion proteins, either sodium channel of the nerve membrane is considered to fused to ribosomal protein or to themselves (Baker and be the primary target of pyrethroid insecticides and DDT, Board, 1991; Mezquita et al., 1997). According to the and nerve insensitivity is one of the major mechanisms different types of ribosome proteins, ubiquitin ribosome * Correspondence: [email protected] 506 ZHANG et al. / Turk J Zool precursor protein genes also can be divided into 2 5’-ATGCAGATYTTCGTNAARACC-3’ and the reverse classes: RpL40 (Uba52) and RpS27a (Uba80). Both types primer 5’-YYATTTGTCGTCRTCYTTGAAG-3’. PCR of translation products are then processed by specific conditions were as follows: initial denaturation at 95 °C for enzymes (Amerik and Hochstrasser, 2004). 3 min, followed by 30 cycles at 95 °C for 30 s, 45 °C for 30 To obtain the deltamethrin-resistance–associated s, and 72 °C for 1 min, with a final 72 °C extension for 10 genes, we isolated and characterized the differentially min. cDNA fragments were separated by electrophoresis expressed ubiquitin gene by cDNA representational on a 1% agarose gel, and PCR products were purified difference analysis and found that the ubiquitin protein using a Nucleic Acid Purification Kit (Axygen, USA). The is differentially expressed in the deltamethrin-resistant purified products were ligated into a pMD 19-T vector and -susceptible strains of P. xylostella (Cheng et al., 2005; (TaKaRa) at 16 °C for 30 min, and then the ligation mixture Cheng et al., 2009). Because there are 3 types of ubiquitin was transformed into E. coli DH5α competent cells and gene, we isolated and sequenced the complete coding cultured on LB plates containing ampicillin (100 mg/ sequence of 1 type of ubiquitin gene (ubiquitin ribosome mL), IPTG (24 mg/mL), and X-Gal (20 mg/mL). White fusion gene RpS27a) from diamondback moth. The colonies were picked out and subjected to colony PCR to expression pattern of RpS27a in the fourth instar larvae confirm the presence of the expected band. Plasmid DNA of the 2 diamondback moth strains was characterized, and was extracted using the AxyPrep Plasmid Miniprep Kit its possible correlation with pesticide resistance was also (Axygen) and sequenced by an automated DNA sequencer discussed. (Applied Biosystems, USA). 2.4. Sequence analysis of RpS27a gene 2. Materials and methods The amino acid sequence, protein molecular mass, and 2.1. Plutella xylostella isoelectric point (pI) of RpS27a were predicted with the P. xylostella was obtained from Huaxi cabbage fields Compute pI/Mw tool (http://www.expasy.org/). The (Guiyang, China). Toxicity evaluation showed that it was potential subcellular localization was predicted on the a deltamethrin-susceptible strain (LD50 = 0.55 µg/larva). online server (http://psort.hgc.jp/form2.html). Homology It was reared in our laboratory as the standard susceptible search was performed with the BLAST program at the strain in this study. National Center for Biotechnology Information. Multiple The deltamethrin-resistant strain of P. xylostella was sequence alignment was performed with the Clustal X established and maintained by our laboratory selection program (Jeanmougin, 1998). A phylogenetic tree was (Liu et al., 1995), with continuous exposure to deltamethrin constructed using MEGA 4.0 programs (Tamura et al., (Roussel-Uclaf, France) of the susceptible strain generation 2007) using the neighbor-joining method. Bootstrap by generation as previously described (FAO, 1979). After values were calculated on 1000 replications. more than 300 generations of laboratory selection, the 2.5. Quantitative real-time PCR analysis resistance level was increased over 5000 times compared The relative transcription levels of RpS27a in the fourth with the susceptible strain. Larvae of both strains were instar larvae of P. xylostella from the deltamethrin- kept at 26 ± 1 °C with a constant photoperiod of 16 h light resistant and -susceptible strains were examined by and 8 h dark and 75% relative humidity. quantitative real-time PCR (qRT-PCR). Total RNAs 2.2. Total RNA extraction and cDNA synthesis were extracted from both strains using the RNeasy Mini Total RNA of 10 whole larvae was extracted from fourth Kit (QIAGEN). The purity and concentration of total instar larvae of both the deltamethrin-resistant and RNAs were determined by spectrophotometry. The RNA -susceptible strains using the RNeasy Mini Kit (QIAGEN, integrity was observed on a 1.5% agarose gel. RNA (1 Germany) according to the manufacturer’s instructions. µg) was synthesized into cDNA. Real-time PCR was To avoid potential DNA contamination, total RNA was conducted using the LightCycler real-time PCR detection treated with DNase I (TaKaRa, Japan). Total RNA was system (Roche, Switzerland) with the housekeeping gene subjected to gel electrophoresis after extraction. RNA GAPDH as the internal control. Two pairs of qRT-PCR concentration and purity were measured with a NanoDrop specific primers were designed using Primer Premier 1000 spectrophotometer (Thermo, USA). The first strand 5 software (Lalitha, 2000): RpS27a gene (forward: 5’- cDNA was synthesized with the PrimeScript RT Reagent TCGCACGCTCTCCGACTACAAC-3’; reverse: Kit (TaKaRa) following the manufacturer’s protocol. 5’-CAACCTTGTAGAACCTGAGCACGG-3’) and 2.3. Cloning and sequencing
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