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cDNA cloning and characterization of the carboxylesterase pxCCE016b from the diamondback moth, Plutella xylostella L.
Journal of Integrative Agriculture 2016, 15(5): 1059–1068 Available online at www.sciencedirect.com
ScienceDirect
RESEARCH ARTICLE
cDNA cloning and characterization of the carboxylesterase pxCCE016b from the diamondback moth, Plutella xylostella L. HU Zhen-di1, 2, FENG Xia2, LIN Qing-sheng2, CHEN Huan-yu2, LI Zhen-yu2, YIN Fei2, LIANG Pei1, GAO Xi-wu1 1
Department of Entomology, China Agricultural University, Beijing 100193, P.R.China
2
Institute of Plant Protection/Guangdong Provincial Key Laboratory of High Technology for Plant Protection, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, P.R.China
Abstract Carboxylesterase is a multifunctional superfamily and can be found in almost all living organisms. As the metabolic enzymes, carboxylesterases are involved in insecticides resistance in insects for long time. In our previous studies, the enhanced carboxylesterase activities were found in the chlorantraniliprole resistance strain of diamondback moth (DBM). However, the related enzyme gene of chlorantraniliprole resistance has not been clear in this strain. Here, a full-length cDNA of carboxylesterase pxCCE016b was cloned and exogenously expressed in Escherichia coli at the first time, which contained a 1 693 bp open reading frame (ORF) and encoded a protein of 542 amino acids. Sequence analysis showed that this cDNA has a predicted mass of 61.56 kDa and a theoretical isoelectric point value of 5.78. The sequence of deduced amino acid possessed the classical structural features: a type-B carboxylesterase signature 2 (EDCLYLNVYTK), a type-B carboxylesterase serine active site (FGGDPENITIFGESAG) and the catalytic triad (Ser186, Glu316, and His444). The real-time quantitative PCR (qPCR) analysis showed that the expression level of the pxCCE016b was significantly higher in the chlorantraniliprole resistant strain than in the susceptible strain. Furthermore, pxCCE016b was highly expressed in the midgut and epidermis of the DBM larvae. When the 3rd-instar larvae of resistant DBM were exposed to abamectin, alpha-cypermethrin, chlorantraniliprole, spinosad, chlorfenapyr and indoxacarb insecticides, the up-regulated expression of pxCCE016b was observed only in the group treated by chlorantraniliprole. In addition, recombinant vector pET-pxCCE016b was constructed with the most coding region (1 293 bp) and large number of soluble recombinant proteins (less than 48 kDa) were expressed successfully with prokaryotic cell. Western blot analysis showed that it was coded by pxCCE016b. All the above findings provide important information for further functional study, although we are uncertainty whether the pxCCE016b gene is actually involved in chlorantraniliprole resistance. Keywords: Plutella xylostella, carboxylesterase, chlorantraniliprole, insecticide resistance, pxCCE016b
Received 20 August, 2015 Accepted 24 December, 2015 HU Zhen-di, E-mail: [email protected]; Correspondence GAO Xi-wu, Tel: +86-10-62732974, Fax: +86-10-62731306, E-mail: [email protected] © 2016, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(15)61278-3
1. Introduction The diamondback moth (DBM), Plutella xylostella (L.), is one of the most important cruciferous pests in China (Li et al. 2016) and around the world (Talekar and Shelton 1993). DBM cost the world economy about 4–5 billion USD per
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year in losses (Furlong et al. 2013). It is well known that DBM has developed various levels of resistance to all major classes of insecticides used (Zhao et al. 2002; Heckel et al. 2004; Sayyed and Wright 2006; Whalon et al. 2008). As the first commercial insecticide of new anthranilic diamide class, the chlorantraniliprole was applied as an ideal reduced-risk rotation insecticide for insecticide resistance management (IRM) program (Hannig et al. 2009; Silva et al. 2012). In China, chlorantraniliprole was recommended as an effective rotation insecticide for the control of DBM since 2008. However, DBM developed highly resistance to this insecticide in the field populations in Guangdong Province because of the intensive usage during 3–5 years (Wang et al. 2010; Wang and Wu 2012). The similar situation has happened in Philippine Islands (Edralin et al. 2011). In order to delay resistance evolution in the field, it is important to study the relative resistance mechanisms of DBM. The major types of mechanisms, by which DBM become resistant to insecticides, are frequently associated with increased activities of detoxifying enzymes, reduced target site or nerve sensitivity, and decreased cuticular penetration (Eziah et al. 2009). The target site mutation resistance has been shown to be involved in resistance to chlorantraniliprole (Troczka et al. 2012). In addition, through synergism of enzyme inhibitors experiment, enzyme activities analysis and biochemical response test to insecticide-induced stress, detoxifying enzymes dependent resistance is observed in resistant strains of DBM from China (Wang and Wu et al. 2012; Hu et al. 2014a). Our previous studies on the transcriptome analysis in DBM also suggested that detoxifying enzymes were the major metabolic factors responsible for chlorantraniliprole resistance (Lin et al. 2013). Based on transcriptome analysis, we identified a novel P450 gene: CYP321E1 (GenBank accession no. KC 626090) from DBM, RNA interference (RNAi) indicated that this gene was related to chlorantraniliprole resistance (Hu et al. 2014b). Meanwhile, an up-regulation carboxylesterase gene cDNA fragment: Unigene35058_yong_A, was also obtained from DBM EST database. The tag-based digital gene expression (DGE) analysis gave us an opinion that this gene may also play a role in chlorantraniliprole resistance of DBM (Lin et al. 2013). Esterase is one of the major detoxifying enzymes in insects, insecticide resistance mediated by which has been found in many different insects (Zhang et al. 2010). As reported, the mainly molecular basis of this kind of resistance mechanism to some normal used insecticides is gene amplification, up-regulation or main coding sequence mutation (Li et al. 2007). Among them, up-regulation of esterase genes has been found in Myzus persicae, Nilaparvata lugens and Aphis gossypii (Small and Hemingway 2000; Bizzaro et al. 2005; Cao et al. 2008). In DBM, no relative
resistant gene has been cloned and characterized although studies suggested that carboxylesterases may be involved in chlorantraniliprole resistance. In this paper, a full-length cDNA of pxCCE016b from DBM was identified and analyzed. Expression pattern of pxCCE016b and exposure experiments to several normal used insecticides were investigated. In addition, recombinant protein of pxCCE016b cDNA was expressed in Escherichia coli and then Western blot analysis was conducted. Our findings here provide important information for further functional study, although we are uncertainty whether the pxCCE016b is actually involved in chlorantraniliprole.
2. Results 2.1. cDNA cloning of pxCCE016b Using the specific primers, the core sequence of pxCCE016b about 600 bp was obtained firstly. Based on it, 5´/3´ rapid-amplification of cDNA ends (RACE) PCR reactions were performed respectively, and then the full-length cDNA sequence of pxCCE016b gene was obtained by overlapping three fragments. This cDNA is 1 773 bp long (GenBank accession no. KM008609) with an open reading frame (ORF) of 1 693 bp (Fig. 1), encoding 542 amino acids. Analysis of deduced amino acid sequence showed that this cDNA has a molecular weight of 61.56 kDa and a theoretical isoelectric point value of 5.78. But no signal sequence was observed.
2.2. Sequence analysis Sequence analysis indicated that the translated amino acid of pxCCE016b had high homology to type-B carboxylesterases from other lepidopteran insects in the GenBank database (mostly >50%). Among them, pxCCE016b shared 53% identity in its deduced amino acid with that of CCE016b from Helicoverpa armigera (GenBank accession no. ADF43479), but whose function is still unrevealed (Teese et al. 2010). Multi-sequence alignment analysis also showed that the amino acid sequence encoded by the pxCCE016b contains characteristic conservative regions of carboxylesterases: a type-B carboxylesterase signature 2 motif (EDCLYLNVYTK), a type-B carboxylesterase serine motif (FGGDPENITIFGESAG) and the catalytic triad (Ser186, Glu316, and His444). Furthermore, the common sequence GXSXG at the active site of carboxylesterase serine motif was also observed (Fig. 2). Here, the phylogenetic tree, generated by 10 amino acid sequences of different insect carboxylesterases, showed that the current gene was grouped with CCE016 (Fig. 3). Although its overall identity to CCE016b is only 53%, they are still considered to the same family for its conserved sequence in salient characteristics region.
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Fig. 1 Complete nucleotide and deduced amino acid sequence of pxCCE016b cDNA of Plutella xylostella. The type-B carboxylesterase signature 2 motif (EDCLYLNVYTK) and serine motif (FGGDPENITIFGESAG) are highlighted in grey; the catalytic triad are highlighted in black. The sequence was deposited in the GenBank (accession no. KM008609).
ADX30519 KM008609 ADF43479 ABE01157 ABQ59309
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Fig. 2 Comparison of amino acid sequences of pxCCE016b (KM008609) from P. xylostella, carboxylesterase 6 (ADX30519) from P. xylostella, CCE016b (ADF43479) from Helicoverpa armigera, carboxylesterase (ABE01157) from Spodoptera litura, carboxylesterase (ABQ59309) from Spodoptera exigua, several conserved motifs of these carboxylesterase proteins are boxed.
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2.3. Expression profile of pxCCE016b
2.4. Effect of insecticide exposure on pxCCE016b expression
The relative expression level of pxCCE016b gene was determined firstly in the chlorantraniliprole susceptible and resistant strains by using real-time quantitative PCR (qPCR). β-actin was used as an internal reference gene as reported by Kim et al. (2010) and Lin et al. (2013). Compared to the susceptible strain, expression level of pxCCE016b in the resistant strain was significantly high (approximately 49.04 folds, P<0.05) (Fig. 4-A). The tissue-dependent expression of pxCCE016b was further examined in the 4th-instar larvae of the resistant strain. As shown in Fig. 4-B, the relatively high expression of pxCCE016b was observed in the midgut and epidermis, but other tissues expression levels were relatively low.
Although our previous studies suggested that pxCCE016b may be involved in DBM resistance against chlorantraniliprole, it is still important to further estimate the effect of several common used insecticides on the expression of this gene. In the exposure experiment, six insecticides: abamectin, alpha-cypermethrin, chlorantraniliprole, spinosad, chlorfenapyr and indoxacarb were used to induce the expression of pxCCE016b, but only chlorantraniliprole up-regulated the expression of pxCCE016b by 2.89-fold at tested concentration for 24 h (P<0.05) (Fig. 5). DBM larvae exposured to spinosad did not show any effect on the gene expression. However, exposure to abamectin,
Fig. 3 A phylogenetic tree constructed based on the carboxylesterase amino acid sequences from P. xylostella and other different insects. The neighbor-joining tree was constructed with 1 000 bootstrap replicates in MEGA 5.0 and branch numbers refer to bootstrap values. Sequences were retrieved from GenBank database. including: pxCCE, P. xylostella, KM008609; CCE016b, H. armigera, ADF43479; CXE6, P. xylostella, ADX30519; CXE3, Spodoptera littoralis, ACV60230; CCE017a, H. armigera, ADF43483; CXEA4, Locusta migratoria, AGT95755; CCE4B, Bombyx mori, BAI66481; CXE17, S. exigua, ADR64700; CXE20, S. littoralis, ACV60247. B
60 50
*
40 30 20 10 0
RS SS Different strains
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Normalized fold expression
A
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c ED
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c HC MG Different tissues
c FB
Fig. 4 Expression characteristic of pxCCE016b. A, transcript expression of pxCCE016b between different strains. RS means resistant strain, SS means susceptible strain. * on the column indicated significant difference by Student’s t-text (P<0.05). B, expression profile of pxCCE016b in different tissues of the resistant 4th-instar larvae. Tissue-dependent expression pattern is examined in five different tissues including epidermis (ED), malpighian tubule (MT), hemocytes (HC), midgut (MG), and fat bodies (FB). β-actin is used as an internal reference gene. Data are means±SE. Different letters on the columns indicate that the means of the gene transcript levels have significant difference among the different tissues based on Fisher’s LSD multiple comparison test (P<0.05). The same as below.
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alpha-cypermethrin, chlorfenapyr and indoxacarb inhibited pxCCE016b expression at different levels ranging from 0.33 to 0.83 folds (P<0.05).
2.5. Prokaryotic expression of pxCCE016b and Western blot analysis To further investigate the functions of pxCCE016b, the prokaryotic expression of this gene was performed. Under the conditions as described in the section of Materials and methods, a large number of soluble recombinant proteins of pxCCE016b were obtained. The induced products were then identified by SDS-PAGE gel. Compared with the control, there was an induced protein band (less than 48 kDa) on the SDS-PAGE gel, which matched the predicted molecular weight calculated from its major ORF sequence (1 293 bp) (Fig. 6). Meanwhile, the Western blot analysis was carried out to confirm the induced protein. After electrophoresis, the induced products were transferred from SDS-PAGE gels onto a polyvinylidene fluoride (PVDF) membrane, and hybridized by the specific antibodies. As shown in Fig. 7, a target hybridization band was observed in the PVDF membrane. However, the control hadn’t hybridization band under the same conditions.
3. Discussion
3
2
1
63 48 35 25 20
Fig. 6 SDS-PAGE of products expressed by pET-pxCCE016b. 1, protein marker; 2, Escherichia coli DE3 containing pETpxCCE016b not induced with isopropl-D-thiog-alactoside (IPTG); 3, E. coli DE3 containing pET-pxCCE016b induced with IPTG.
1
2
3
kDa 100 75
Carboxylesterases are involved in a wide range of functions, such as insecticides detoxification, developmental regulation or sex pheromone degradation. Insect carboxylesterases usually are considered as a kind of metabolic enzymes, 3.5
a
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48
35
3.0 2.5 2.0 1.5
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b
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c
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K C
rb ca
py r
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sa d
or fe na
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in o
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ra
C
hl o
ra nt
-m et
ph a Al
Ab
hr in
tin
0
am ec
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kDa 245 180 135 100 75
Different insecticides
Fig. 5 Effect of abamectin, alpha-cypermethrin, chlorantraniliprole, spinosad, chlorfenapyr and indoxacarb on the expression of pxCCE016b after the larvae of P. xylostella treated with each insecticide at the dose of about (lethal concentration 30 (LC30) (20, 500, 50, 5, 100 and 40 μg mL–1, respectively), and analyzed at 24 h by qPCR. Sterilized water was as control (CK).
Fig. 7 Western blot analysis result. 1, the plasmid pETpxCCE016b expresses the target protein (induce with IPTG); 2, the plasmid pET-pxCCE016b do not express the protein (not induced with IPTG, as control); 3, protein marker.
which often implicates in the resistance to a variety of insecticides. To date, many carboxylesterases genes, which were considered relating with insecticides resistance, have been isolated from various insects (Oakeshott et al. 1993, 2005; Ranson et al. 2002; Claudianos et al. 2006; Strode
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et al. 2008; Teese et al. 2010). As reported, most insect carboxylesterases are characterized as members of the carboxyl/cholinesterase (CCE) superfamily. Structurally CCEs contain a conserved catalytic triad: a serine (normally surrounded by the conserved common sequence GXSXG), a glutamate and a histidine (Ollis et al. 1992). However, few CCE genes from DBM have been isolated and reported to be involved in chlorantraniliprole resistance. Here, the fulllength cDNA of pxCCE016b was isolated from DBM for the first time, and analysis of deduced amino acid sequence showed it shares all salient characteristics of carboxylesterases. Comparative transcript expression between different DBM strains by qPCR revealed higher expression level of pxCCE016b in the resistant strain than the susceptible one. The above findings are consistent well with our previous studies (Lin et al. 2013). Yu et al. (2009) proposed that differential tissue-dependent expression patterns of insect detoxifying enzyme genes may imply their different functions. Here, the expression pattern of pxCCE016b gene in different tissues of the resistant 4th-instar larvae was also analyzed by qPCR. The results suggest that this gene expression was the highest in midgut, followed by epidermis. The insect midgut, as the barrier of stomach toxicant, is commonly associated with digestive and detoxification functions (Cohen et al. 1992; Snyder et al. 1995; Yu et al. 2003). And several CCEs are proposed to have these functions based on their expression in the insect midgut (Oakeshott et al. 1993). As a stomach and contact insecticide, chlorantraniliprole enters DBM larvae by eating and touching, suggesting that higher expressions of detoxifying enzyme genes in midgut and epidermis may be coincided well with these tissues functions. The findings in this paper are in agreement with our initial concept about pxCCE016b expression characteristic. The adaptive regulation ability of insect carboxylesterases in response to various environmental changes over the course of development, especially to an exposure to chemicals, has been deeply studied (Hosokawa et al. 1988; Satoh and Hosokawa 2006). Here, in order to examine the putative detoxification function of pxCCE016b, the 3rd-instar larvae of DBM were exposed to six common used insecticides. The results showed that the expression of pxCCE016b only could be up-regulated by chlorantraniliprole. It indicated that pxCCE016b might play some role in detoxification of DBM against chlorantraniliprole.
4. Conclusion In summary, the full-length cDNA of pxCCE016b was isolated and cloned from DBM successfully. Sequence characteristics studies showed this gene belongs to the CCEs family. Expression characteristics researches carried
out here suggested that the overexpression of pxCCE016b might be one of resistance mechanisms of DBM to chlorantraniliprole, but it is not involved in resistance produce for spinosad, abamectin, chlorfenapyr, alpha-cypermethrin and indoxacarb. All of the results here also provided molecular basis for further gene functional studies of the pxCCE016b, which will help us to better understand the DBM resistance mechanisms.
5. Materials and methods 5.1. Insects Two DBM stains, the resistance to chlorantraniliprole and the other sensitive to this insecticide, were used for this study. The resistant strain was established in 2012 from a cabbage field population collected in Guangxi Province, China. The strain was continuously selected with chlorantraniliprole every two to three generations to get rid of the influence factors (Lin et al. 2013), and the resistant ration was about 80. The susceptible strain, which was friendly provided by the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, was collected in 2007 at Shenzhen, China and has never been exposured to any chemicals. Both DBM stains were reared in the laboratory with the conditions described by Hu et al. (2014b). The DBM larvae and adults were fed on fresh leaves of Brassica campestris L. and 10% honey solution, respectively.
5.2. Extraction of total RNA and synthesis of cDNA The whole 4th-instar larvae of the resistant DBM strain were used to extract total RNA with Easy Spin Total RNA Extraction Kit (Biomed, China). The first-strand cDNA and 3´-/5´-ends RACE cDNAs were synthesized following the manufacturer’s instructions of the Reverse Transcriptase M-MLV (TaKaRa, China) and SMARTTM RACE cDNA Amplification (Clonthch, USA), respectively. The total RNA was making an assay of the absorbance by NANO-200 ultramicro nucleic acid analyzer at 260 nm, and then checked the quality by 1.2% agarose gels electrophoresis. The obtained cDNAs were stored at –80°C for our further experiments.
5.3. Cloning of pxCCE016b cDNA To clone the full-length cDNA sequence of pxCCE016b, a pair of primers (5´-ACG TGC GAG ATT TCT TGC TT-3´ and 5´-AAT TCC GTA TGC TGC TCC AC-3´) were designed at first for the core fragment amplification. The obtained core sequence was then used as template to design gene-specific primers (GSPs) for 3´-/5´-ends RACE PCR. The information of GSPs were as the following: 5´-end RACE, CAR-GSP1
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(5´-CTG GTG GCA ATT AGG CCC ATG CTT TA-3´, used in the first-round PCR) and CAR-NGSP1 (5´-CCA CTC CAT GGT TTA AGA GGC TGC GG-3´, used in the second-round PCR); 3´-end RACE: CYP-GSP2 (5´-GGC TAA GCG CCC TGA CAT ACT AAA AC-3´, used in the first-round PCR) and CYP-NGSP2 (5´-CGT TTA CAT CCA CCG ATT TCG CTG CT-3´, used in the second-round PCR). The mixed universal primer (long: 5´-CTA ATA CGA CTC ACT ATA GGG CAA GCA GTG GTA TCA ACG CAG AGT-3´; short: 5´-CTA ATA CGA CTC ACT ATA GGG C-3´) was used as the reverse primer. The steps of PCR were: 1 cycle at 94°C for 5 min; 35 cycles at 94°C for 1 min, 56°C for 30 s and 72°C for 1 min; 1 cycle at 72°C for 10 min. The PCR products were retrieved, cloned and sequenced, respectively. Then assembled the fragments of each reaction and the full-length cDNA sequence of target gene was obtained. Finally, to confirm the assembled sequence of target gene was correct, the entire ORF region was amplified with the specific primers as following: CAR F (5´-ATG AAA ATG GTG ATA CCA GGC AGT G-3´) and CAR R (5´-TTA AAA ACT CAC TAA TAT GAG GAT T-3´).
5.4. Sequence analysis A comparison of pxCCE016b cDNA with other esterase sequences (deposit in GenBank) was performed using online Blast tool (http://www.ncbi.nlm.nih.gov). The translation of pxCCE016b was carried out with ExPASy (http://www. expasy.org/tools). According to the deduced amino acid sequence, a phylogenetic tree was constructed with MEGA 5.0 software. And then a multiple sequence alignment was carried out using Multiple Alignment software (http://www. phylogeny.fr/). Other protein analyses were also performed with ExPASy (http://www.expasy.org/tools), such as a theoretical isoelectric point (pI) and a predicted molecular weight.
5.5. Expression pattern of pxCCE016b The relative expression levels of pxCCE016b in the 4th-instar larvae of susceptible and resistant DBM strains were detected using qPCR firstly. For the tissue-dependent expression pattern analysis, five different tissues of the 4th-instar larvae: epidermis, fat bodies, hemocytes, midgut and malpighian tubule, were collected for total RNA extraction, respectively. Then the first-strand cDNA for qPCR analysis was obtained with iScriptTM cDNA Synthesis Kit (Bio-Rad, USA). All the experiments were independently repeated three times with different RNA preparations. The total RNA and synthesized cDNA were also quantified by performing the absorbance at 260 nm (NANO-200 ultramicro nucleic acid analyzer), respectively. And then stored at –20°C for qPCR analysis.
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All qPCR reactions were carried out on the CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, USA), the test procedures were reference to our previous study (Hu et al. 2014b). Specific primers were designed based on the full-length cDNA of pxCCE016b, the detail information were as following: CarE-q-PCR-F, 5´-ACT TGG AAG CAC CTG GC-3´ and CarE-q-PCR-R, 5´-CTC CAG CAC TTT CAC CA-3´. The housekeeping gene β-actin of DBM was used as a reference according to Kim et al. (2010). And specific primers for DBM β-actin gene were FP 5´-TGG CAC ACC TTC TAC-3´ and RP 5´-CAT GAT CTG GGT CAT CTT CT-3´ as reported by Lin et al. ( 2013).
5.6. Insecticide exposure experiments Six common used insecticides: abamectin, alpha-cypermethrin, chlorantraniliprole, spinosad, chlorfenapyr and indoxacarb, were used to evaluate the effect on expression of pxCCE016b in resistant DBM larvae. For exposure test, the leaf dip method reported by Tabashnik et al. (1987) was used, and the cabbage leaf discs were dipped into different insecticide solutions containing 0.05% triton X-100 at the about LC30 dose (20, 500, 50, 5, 100 and 40 μg mL–1, respectively). The 3rd-instar larvae of DBM were fed with cabbage leaf discs coated with different insecticides for 24 h. The larvae treated with the sterilized water with 0.05% triton X-100 were used as the control. Each treatment was repeated three times. After 24 h exposure, the live larvae were collected and used for further experiments. The methods applied for total RNA extraction, first-strand cDNA synthesis and qPCR analysis were as same as described above.
5.7. Prokaryotic expression of pxCCE016b gene Based on the specific primers, ORF-F (5´-GCGGATCC ATG AAA ATG GTG ATA CCA GGC-3´) and ORF-R (5´-CCCAAGCTT ATA GAC AAG AGC AAA CCT AGC3´), the most coding region of pxCCE016b cDNA was obtained. The PCR product was retrieved and purified, and then inserted into the pMD18-T vector to construct the pMD18-pxCCE016b plasmid. After digested with restriction endonucleases BamHI and HindIII enzymes, the target fragment was ligated with T4 DNA ligase into the expression vector pET-32a(+) (Tiangen, China), which was also digested by the same restriction endonucleases enzymes before ligation. The construction diagram sees Fig. 8. The constructed plasmid solution was transformed into BL21 (DE3) competent cells (TaKaRa, Japan). After the positive colonies were selected and identified by restriction endonucleases BamHI and HindIII enzymes, the accuracy of the expressed plasmid was also verified by sequencing again. Subsequently, the BL21 (DE3) competent cells containing
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+
HindIII (173)
pET-pxCCE016b pxCCE016b
National Natural Science Foundation of China (31501664), the President Foundation of Guangdong Academy of Agricultural Sciences, China (201514), the Science and Technology Planning Project of Guangdong Province, China (2013B050800019 and 2014B070706017) and the Agro-Scientific Research Special Fund in the Public Interest, China (201103021).
References
T7 promoter
BamHI (198)
Fig. 8 Construction diagram of the pET-pxCCE016b.
recombinant plasmid was cultivated in Lucia-Bertani (LB) liquid medium and added the isopropl-D-thiog-alactoside (IPTG) to terminal concentration of 0.6 mmol L–1. Harvested the bacteria cells after 4–6 h cultivation with shaking and confirmed the expected fusion protein by SDS-PAGE eletrophoresis.
5.8. Western blot analysis Once it has been confirmed that the expected protein could be expressed in the BL21 (DE3) cells, Western blot analysis was applied to further identify of expression. The BL21 (DE3) cells contained the pET-pxCCE016b plasmid was induced under the same condition as prokaryotic expression experiment. The expressed bacteria were collected by centrifugation and suspension again with phosphate buffered saline (PBS). After added with 5×SDS-PAGE loading buffer (contains β-mercaptoethanol, Tiangen, China), the harvested bacteria cells were denatured at 100°C for 8–10 min and 10 μL of products per land was loaded on SDS-PAGE gel for electrophoresis analysis. Subsequently, the expression products were transferred from SDS-PAGE gel to a PVDF membrane by the Trans-Blot SD Semi-Dry Electrophoretic Transfer cell (Bio-Rad, USA) at 20 V for 15 min. The following Western blot analysis was carried out according to the reported procedures (Feng et al. 2012). When the PVDF membrane was incubated, the anti His-Tag mouse monoclonal antibody and affinipure goat anti-mouse IgG (H+L) were used as the primary and secondary antibody respectively. Finally, the color reaction of the membrane was carried out with DAB Kit (Chaorui, China).
Acknowledgements This study was funded by following research programs: the
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RESEARCH ARTICLE
cDNA cloning and characterization of the carboxylesterase pxCCE016b from the diamondback moth, Plutella xylostella L. HU Zhen-di1, 2, FENG Xia2, LIN Qing-sheng2, CHEN Huan-yu2, LI Zhen-yu2, YIN Fei2, LIANG Pei1, GAO Xi-wu1 1
Department of Entomology, China Agricultural University, Beijing 100193, P.R.China
2
Institute of Plant Protection/Guangdong Provincial Key Laboratory of High Technology for Plant Protection, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, P.R.China
Abstract Carboxylesterase is a multifunctional superfamily and can be found in almost all living organisms. As the metabolic enzymes, carboxylesterases are involved in insecticides resistance in insects for long time. In our previous studies, the enhanced carboxylesterase activities were found in the chlorantraniliprole resistance strain of diamondback moth (DBM). However, the related enzyme gene of chlorantraniliprole resistance has not been clear in this strain. Here, a full-length cDNA of carboxylesterase pxCCE016b was cloned and exogenously expressed in Escherichia coli at the first time, which contained a 1 693 bp open reading frame (ORF) and encoded a protein of 542 amino acids. Sequence analysis showed that this cDNA has a predicted mass of 61.56 kDa and a theoretical isoelectric point value of 5.78. The sequence of deduced amino acid possessed the classical structural features: a type-B carboxylesterase signature 2 (EDCLYLNVYTK), a type-B carboxylesterase serine active site (FGGDPENITIFGESAG) and the catalytic triad (Ser186, Glu316, and His444). The real-time quantitative PCR (qPCR) analysis showed that the expression level of the pxCCE016b was significantly higher in the chlorantraniliprole resistant strain than in the susceptible strain. Furthermore, pxCCE016b was highly expressed in the midgut and epidermis of the DBM larvae. When the 3rd-instar larvae of resistant DBM were exposed to abamectin, alpha-cypermethrin, chlorantraniliprole, spinosad, chlorfenapyr and indoxacarb insecticides, the up-regulated expression of pxCCE016b was observed only in the group treated by chlorantraniliprole. In addition, recombinant vector pET-pxCCE016b was constructed with the most coding region (1 293 bp) and large number of soluble recombinant proteins (less than 48 kDa) were expressed successfully with prokaryotic cell. Western blot analysis showed that it was coded by pxCCE016b. All the above findings provide important information for further functional study, although we are uncertainty whether the pxCCE016b gene is actually involved in chlorantraniliprole resistance. Keywords: Plutella xylostella, carboxylesterase, chlorantraniliprole, insecticide resistance, pxCCE016b
Received 20 August, 2015 Accepted 24 December, 2015 HU Zhen-di, E-mail: [email protected]; Correspondence GAO Xi-wu, Tel: +86-10-62732974, Fax: +86-10-62731306, E-mail: [email protected] © 2016, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(15)61278-3
1. Introduction The diamondback moth (DBM), Plutella xylostella (L.), is one of the most important cruciferous pests in China (Li et al. 2016) and around the world (Talekar and Shelton 1993). DBM cost the world economy about 4–5 billion USD per
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year in losses (Furlong et al. 2013). It is well known that DBM has developed various levels of resistance to all major classes of insecticides used (Zhao et al. 2002; Heckel et al. 2004; Sayyed and Wright 2006; Whalon et al. 2008). As the first commercial insecticide of new anthranilic diamide class, the chlorantraniliprole was applied as an ideal reduced-risk rotation insecticide for insecticide resistance management (IRM) program (Hannig et al. 2009; Silva et al. 2012). In China, chlorantraniliprole was recommended as an effective rotation insecticide for the control of DBM since 2008. However, DBM developed highly resistance to this insecticide in the field populations in Guangdong Province because of the intensive usage during 3–5 years (Wang et al. 2010; Wang and Wu 2012). The similar situation has happened in Philippine Islands (Edralin et al. 2011). In order to delay resistance evolution in the field, it is important to study the relative resistance mechanisms of DBM. The major types of mechanisms, by which DBM become resistant to insecticides, are frequently associated with increased activities of detoxifying enzymes, reduced target site or nerve sensitivity, and decreased cuticular penetration (Eziah et al. 2009). The target site mutation resistance has been shown to be involved in resistance to chlorantraniliprole (Troczka et al. 2012). In addition, through synergism of enzyme inhibitors experiment, enzyme activities analysis and biochemical response test to insecticide-induced stress, detoxifying enzymes dependent resistance is observed in resistant strains of DBM from China (Wang and Wu et al. 2012; Hu et al. 2014a). Our previous studies on the transcriptome analysis in DBM also suggested that detoxifying enzymes were the major metabolic factors responsible for chlorantraniliprole resistance (Lin et al. 2013). Based on transcriptome analysis, we identified a novel P450 gene: CYP321E1 (GenBank accession no. KC 626090) from DBM, RNA interference (RNAi) indicated that this gene was related to chlorantraniliprole resistance (Hu et al. 2014b). Meanwhile, an up-regulation carboxylesterase gene cDNA fragment: Unigene35058_yong_A, was also obtained from DBM EST database. The tag-based digital gene expression (DGE) analysis gave us an opinion that this gene may also play a role in chlorantraniliprole resistance of DBM (Lin et al. 2013). Esterase is one of the major detoxifying enzymes in insects, insecticide resistance mediated by which has been found in many different insects (Zhang et al. 2010). As reported, the mainly molecular basis of this kind of resistance mechanism to some normal used insecticides is gene amplification, up-regulation or main coding sequence mutation (Li et al. 2007). Among them, up-regulation of esterase genes has been found in Myzus persicae, Nilaparvata lugens and Aphis gossypii (Small and Hemingway 2000; Bizzaro et al. 2005; Cao et al. 2008). In DBM, no relative
resistant gene has been cloned and characterized although studies suggested that carboxylesterases may be involved in chlorantraniliprole resistance. In this paper, a full-length cDNA of pxCCE016b from DBM was identified and analyzed. Expression pattern of pxCCE016b and exposure experiments to several normal used insecticides were investigated. In addition, recombinant protein of pxCCE016b cDNA was expressed in Escherichia coli and then Western blot analysis was conducted. Our findings here provide important information for further functional study, although we are uncertainty whether the pxCCE016b is actually involved in chlorantraniliprole.
2. Results 2.1. cDNA cloning of pxCCE016b Using the specific primers, the core sequence of pxCCE016b about 600 bp was obtained firstly. Based on it, 5´/3´ rapid-amplification of cDNA ends (RACE) PCR reactions were performed respectively, and then the full-length cDNA sequence of pxCCE016b gene was obtained by overlapping three fragments. This cDNA is 1 773 bp long (GenBank accession no. KM008609) with an open reading frame (ORF) of 1 693 bp (Fig. 1), encoding 542 amino acids. Analysis of deduced amino acid sequence showed that this cDNA has a molecular weight of 61.56 kDa and a theoretical isoelectric point value of 5.78. But no signal sequence was observed.
2.2. Sequence analysis Sequence analysis indicated that the translated amino acid of pxCCE016b had high homology to type-B carboxylesterases from other lepidopteran insects in the GenBank database (mostly >50%). Among them, pxCCE016b shared 53% identity in its deduced amino acid with that of CCE016b from Helicoverpa armigera (GenBank accession no. ADF43479), but whose function is still unrevealed (Teese et al. 2010). Multi-sequence alignment analysis also showed that the amino acid sequence encoded by the pxCCE016b contains characteristic conservative regions of carboxylesterases: a type-B carboxylesterase signature 2 motif (EDCLYLNVYTK), a type-B carboxylesterase serine motif (FGGDPENITIFGESAG) and the catalytic triad (Ser186, Glu316, and His444). Furthermore, the common sequence GXSXG at the active site of carboxylesterase serine motif was also observed (Fig. 2). Here, the phylogenetic tree, generated by 10 amino acid sequences of different insect carboxylesterases, showed that the current gene was grouped with CCE016 (Fig. 3). Although its overall identity to CCE016b is only 53%, they are still considered to the same family for its conserved sequence in salient characteristics region.
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Fig. 1 Complete nucleotide and deduced amino acid sequence of pxCCE016b cDNA of Plutella xylostella. The type-B carboxylesterase signature 2 motif (EDCLYLNVYTK) and serine motif (FGGDPENITIFGESAG) are highlighted in grey; the catalytic triad are highlighted in black. The sequence was deposited in the GenBank (accession no. KM008609).
ADX30519 KM008609 ADF43479 ABE01157 ABQ59309
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ADX30519 KM008609 ADF43479 ABE01157 ABQ59309
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Fig. 2 Comparison of amino acid sequences of pxCCE016b (KM008609) from P. xylostella, carboxylesterase 6 (ADX30519) from P. xylostella, CCE016b (ADF43479) from Helicoverpa armigera, carboxylesterase (ABE01157) from Spodoptera litura, carboxylesterase (ABQ59309) from Spodoptera exigua, several conserved motifs of these carboxylesterase proteins are boxed.
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2.3. Expression profile of pxCCE016b
2.4. Effect of insecticide exposure on pxCCE016b expression
The relative expression level of pxCCE016b gene was determined firstly in the chlorantraniliprole susceptible and resistant strains by using real-time quantitative PCR (qPCR). β-actin was used as an internal reference gene as reported by Kim et al. (2010) and Lin et al. (2013). Compared to the susceptible strain, expression level of pxCCE016b in the resistant strain was significantly high (approximately 49.04 folds, P<0.05) (Fig. 4-A). The tissue-dependent expression of pxCCE016b was further examined in the 4th-instar larvae of the resistant strain. As shown in Fig. 4-B, the relatively high expression of pxCCE016b was observed in the midgut and epidermis, but other tissues expression levels were relatively low.
Although our previous studies suggested that pxCCE016b may be involved in DBM resistance against chlorantraniliprole, it is still important to further estimate the effect of several common used insecticides on the expression of this gene. In the exposure experiment, six insecticides: abamectin, alpha-cypermethrin, chlorantraniliprole, spinosad, chlorfenapyr and indoxacarb were used to induce the expression of pxCCE016b, but only chlorantraniliprole up-regulated the expression of pxCCE016b by 2.89-fold at tested concentration for 24 h (P<0.05) (Fig. 5). DBM larvae exposured to spinosad did not show any effect on the gene expression. However, exposure to abamectin,
Fig. 3 A phylogenetic tree constructed based on the carboxylesterase amino acid sequences from P. xylostella and other different insects. The neighbor-joining tree was constructed with 1 000 bootstrap replicates in MEGA 5.0 and branch numbers refer to bootstrap values. Sequences were retrieved from GenBank database. including: pxCCE, P. xylostella, KM008609; CCE016b, H. armigera, ADF43479; CXE6, P. xylostella, ADX30519; CXE3, Spodoptera littoralis, ACV60230; CCE017a, H. armigera, ADF43483; CXEA4, Locusta migratoria, AGT95755; CCE4B, Bombyx mori, BAI66481; CXE17, S. exigua, ADR64700; CXE20, S. littoralis, ACV60247. B
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c HC MG Different tissues
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Fig. 4 Expression characteristic of pxCCE016b. A, transcript expression of pxCCE016b between different strains. RS means resistant strain, SS means susceptible strain. * on the column indicated significant difference by Student’s t-text (P<0.05). B, expression profile of pxCCE016b in different tissues of the resistant 4th-instar larvae. Tissue-dependent expression pattern is examined in five different tissues including epidermis (ED), malpighian tubule (MT), hemocytes (HC), midgut (MG), and fat bodies (FB). β-actin is used as an internal reference gene. Data are means±SE. Different letters on the columns indicate that the means of the gene transcript levels have significant difference among the different tissues based on Fisher’s LSD multiple comparison test (P<0.05). The same as below.
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alpha-cypermethrin, chlorfenapyr and indoxacarb inhibited pxCCE016b expression at different levels ranging from 0.33 to 0.83 folds (P<0.05).
2.5. Prokaryotic expression of pxCCE016b and Western blot analysis To further investigate the functions of pxCCE016b, the prokaryotic expression of this gene was performed. Under the conditions as described in the section of Materials and methods, a large number of soluble recombinant proteins of pxCCE016b were obtained. The induced products were then identified by SDS-PAGE gel. Compared with the control, there was an induced protein band (less than 48 kDa) on the SDS-PAGE gel, which matched the predicted molecular weight calculated from its major ORF sequence (1 293 bp) (Fig. 6). Meanwhile, the Western blot analysis was carried out to confirm the induced protein. After electrophoresis, the induced products were transferred from SDS-PAGE gels onto a polyvinylidene fluoride (PVDF) membrane, and hybridized by the specific antibodies. As shown in Fig. 7, a target hybridization band was observed in the PVDF membrane. However, the control hadn’t hybridization band under the same conditions.
3. Discussion
3
2
1
63 48 35 25 20
Fig. 6 SDS-PAGE of products expressed by pET-pxCCE016b. 1, protein marker; 2, Escherichia coli DE3 containing pETpxCCE016b not induced with isopropl-D-thiog-alactoside (IPTG); 3, E. coli DE3 containing pET-pxCCE016b induced with IPTG.
1
2
3
kDa 100 75
Carboxylesterases are involved in a wide range of functions, such as insecticides detoxification, developmental regulation or sex pheromone degradation. Insect carboxylesterases usually are considered as a kind of metabolic enzymes, 3.5
a
63
48
35
3.0 2.5 2.0 1.5
b
b
b
1.0
c
0.5
c
c
25
K C
rb ca
py r
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sa d
or fe na
C
hl
in o
le Sp
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ra
C
hl o
ra nt
-m et
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hr in
tin
0
am ec
Normalized fold expression
kDa 245 180 135 100 75
Different insecticides
Fig. 5 Effect of abamectin, alpha-cypermethrin, chlorantraniliprole, spinosad, chlorfenapyr and indoxacarb on the expression of pxCCE016b after the larvae of P. xylostella treated with each insecticide at the dose of about (lethal concentration 30 (LC30) (20, 500, 50, 5, 100 and 40 μg mL–1, respectively), and analyzed at 24 h by qPCR. Sterilized water was as control (CK).
Fig. 7 Western blot analysis result. 1, the plasmid pETpxCCE016b expresses the target protein (induce with IPTG); 2, the plasmid pET-pxCCE016b do not express the protein (not induced with IPTG, as control); 3, protein marker.
which often implicates in the resistance to a variety of insecticides. To date, many carboxylesterases genes, which were considered relating with insecticides resistance, have been isolated from various insects (Oakeshott et al. 1993, 2005; Ranson et al. 2002; Claudianos et al. 2006; Strode
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et al. 2008; Teese et al. 2010). As reported, most insect carboxylesterases are characterized as members of the carboxyl/cholinesterase (CCE) superfamily. Structurally CCEs contain a conserved catalytic triad: a serine (normally surrounded by the conserved common sequence GXSXG), a glutamate and a histidine (Ollis et al. 1992). However, few CCE genes from DBM have been isolated and reported to be involved in chlorantraniliprole resistance. Here, the fulllength cDNA of pxCCE016b was isolated from DBM for the first time, and analysis of deduced amino acid sequence showed it shares all salient characteristics of carboxylesterases. Comparative transcript expression between different DBM strains by qPCR revealed higher expression level of pxCCE016b in the resistant strain than the susceptible one. The above findings are consistent well with our previous studies (Lin et al. 2013). Yu et al. (2009) proposed that differential tissue-dependent expression patterns of insect detoxifying enzyme genes may imply their different functions. Here, the expression pattern of pxCCE016b gene in different tissues of the resistant 4th-instar larvae was also analyzed by qPCR. The results suggest that this gene expression was the highest in midgut, followed by epidermis. The insect midgut, as the barrier of stomach toxicant, is commonly associated with digestive and detoxification functions (Cohen et al. 1992; Snyder et al. 1995; Yu et al. 2003). And several CCEs are proposed to have these functions based on their expression in the insect midgut (Oakeshott et al. 1993). As a stomach and contact insecticide, chlorantraniliprole enters DBM larvae by eating and touching, suggesting that higher expressions of detoxifying enzyme genes in midgut and epidermis may be coincided well with these tissues functions. The findings in this paper are in agreement with our initial concept about pxCCE016b expression characteristic. The adaptive regulation ability of insect carboxylesterases in response to various environmental changes over the course of development, especially to an exposure to chemicals, has been deeply studied (Hosokawa et al. 1988; Satoh and Hosokawa 2006). Here, in order to examine the putative detoxification function of pxCCE016b, the 3rd-instar larvae of DBM were exposed to six common used insecticides. The results showed that the expression of pxCCE016b only could be up-regulated by chlorantraniliprole. It indicated that pxCCE016b might play some role in detoxification of DBM against chlorantraniliprole.
4. Conclusion In summary, the full-length cDNA of pxCCE016b was isolated and cloned from DBM successfully. Sequence characteristics studies showed this gene belongs to the CCEs family. Expression characteristics researches carried
out here suggested that the overexpression of pxCCE016b might be one of resistance mechanisms of DBM to chlorantraniliprole, but it is not involved in resistance produce for spinosad, abamectin, chlorfenapyr, alpha-cypermethrin and indoxacarb. All of the results here also provided molecular basis for further gene functional studies of the pxCCE016b, which will help us to better understand the DBM resistance mechanisms.
5. Materials and methods 5.1. Insects Two DBM stains, the resistance to chlorantraniliprole and the other sensitive to this insecticide, were used for this study. The resistant strain was established in 2012 from a cabbage field population collected in Guangxi Province, China. The strain was continuously selected with chlorantraniliprole every two to three generations to get rid of the influence factors (Lin et al. 2013), and the resistant ration was about 80. The susceptible strain, which was friendly provided by the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, was collected in 2007 at Shenzhen, China and has never been exposured to any chemicals. Both DBM stains were reared in the laboratory with the conditions described by Hu et al. (2014b). The DBM larvae and adults were fed on fresh leaves of Brassica campestris L. and 10% honey solution, respectively.
5.2. Extraction of total RNA and synthesis of cDNA The whole 4th-instar larvae of the resistant DBM strain were used to extract total RNA with Easy Spin Total RNA Extraction Kit (Biomed, China). The first-strand cDNA and 3´-/5´-ends RACE cDNAs were synthesized following the manufacturer’s instructions of the Reverse Transcriptase M-MLV (TaKaRa, China) and SMARTTM RACE cDNA Amplification (Clonthch, USA), respectively. The total RNA was making an assay of the absorbance by NANO-200 ultramicro nucleic acid analyzer at 260 nm, and then checked the quality by 1.2% agarose gels electrophoresis. The obtained cDNAs were stored at –80°C for our further experiments.
5.3. Cloning of pxCCE016b cDNA To clone the full-length cDNA sequence of pxCCE016b, a pair of primers (5´-ACG TGC GAG ATT TCT TGC TT-3´ and 5´-AAT TCC GTA TGC TGC TCC AC-3´) were designed at first for the core fragment amplification. The obtained core sequence was then used as template to design gene-specific primers (GSPs) for 3´-/5´-ends RACE PCR. The information of GSPs were as the following: 5´-end RACE, CAR-GSP1
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(5´-CTG GTG GCA ATT AGG CCC ATG CTT TA-3´, used in the first-round PCR) and CAR-NGSP1 (5´-CCA CTC CAT GGT TTA AGA GGC TGC GG-3´, used in the second-round PCR); 3´-end RACE: CYP-GSP2 (5´-GGC TAA GCG CCC TGA CAT ACT AAA AC-3´, used in the first-round PCR) and CYP-NGSP2 (5´-CGT TTA CAT CCA CCG ATT TCG CTG CT-3´, used in the second-round PCR). The mixed universal primer (long: 5´-CTA ATA CGA CTC ACT ATA GGG CAA GCA GTG GTA TCA ACG CAG AGT-3´; short: 5´-CTA ATA CGA CTC ACT ATA GGG C-3´) was used as the reverse primer. The steps of PCR were: 1 cycle at 94°C for 5 min; 35 cycles at 94°C for 1 min, 56°C for 30 s and 72°C for 1 min; 1 cycle at 72°C for 10 min. The PCR products were retrieved, cloned and sequenced, respectively. Then assembled the fragments of each reaction and the full-length cDNA sequence of target gene was obtained. Finally, to confirm the assembled sequence of target gene was correct, the entire ORF region was amplified with the specific primers as following: CAR F (5´-ATG AAA ATG GTG ATA CCA GGC AGT G-3´) and CAR R (5´-TTA AAA ACT CAC TAA TAT GAG GAT T-3´).
5.4. Sequence analysis A comparison of pxCCE016b cDNA with other esterase sequences (deposit in GenBank) was performed using online Blast tool (http://www.ncbi.nlm.nih.gov). The translation of pxCCE016b was carried out with ExPASy (http://www. expasy.org/tools). According to the deduced amino acid sequence, a phylogenetic tree was constructed with MEGA 5.0 software. And then a multiple sequence alignment was carried out using Multiple Alignment software (http://www. phylogeny.fr/). Other protein analyses were also performed with ExPASy (http://www.expasy.org/tools), such as a theoretical isoelectric point (pI) and a predicted molecular weight.
5.5. Expression pattern of pxCCE016b The relative expression levels of pxCCE016b in the 4th-instar larvae of susceptible and resistant DBM strains were detected using qPCR firstly. For the tissue-dependent expression pattern analysis, five different tissues of the 4th-instar larvae: epidermis, fat bodies, hemocytes, midgut and malpighian tubule, were collected for total RNA extraction, respectively. Then the first-strand cDNA for qPCR analysis was obtained with iScriptTM cDNA Synthesis Kit (Bio-Rad, USA). All the experiments were independently repeated three times with different RNA preparations. The total RNA and synthesized cDNA were also quantified by performing the absorbance at 260 nm (NANO-200 ultramicro nucleic acid analyzer), respectively. And then stored at –20°C for qPCR analysis.
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All qPCR reactions were carried out on the CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, USA), the test procedures were reference to our previous study (Hu et al. 2014b). Specific primers were designed based on the full-length cDNA of pxCCE016b, the detail information were as following: CarE-q-PCR-F, 5´-ACT TGG AAG CAC CTG GC-3´ and CarE-q-PCR-R, 5´-CTC CAG CAC TTT CAC CA-3´. The housekeeping gene β-actin of DBM was used as a reference according to Kim et al. (2010). And specific primers for DBM β-actin gene were FP 5´-TGG CAC ACC TTC TAC-3´ and RP 5´-CAT GAT CTG GGT CAT CTT CT-3´ as reported by Lin et al. ( 2013).
5.6. Insecticide exposure experiments Six common used insecticides: abamectin, alpha-cypermethrin, chlorantraniliprole, spinosad, chlorfenapyr and indoxacarb, were used to evaluate the effect on expression of pxCCE016b in resistant DBM larvae. For exposure test, the leaf dip method reported by Tabashnik et al. (1987) was used, and the cabbage leaf discs were dipped into different insecticide solutions containing 0.05% triton X-100 at the about LC30 dose (20, 500, 50, 5, 100 and 40 μg mL–1, respectively). The 3rd-instar larvae of DBM were fed with cabbage leaf discs coated with different insecticides for 24 h. The larvae treated with the sterilized water with 0.05% triton X-100 were used as the control. Each treatment was repeated three times. After 24 h exposure, the live larvae were collected and used for further experiments. The methods applied for total RNA extraction, first-strand cDNA synthesis and qPCR analysis were as same as described above.
5.7. Prokaryotic expression of pxCCE016b gene Based on the specific primers, ORF-F (5´-GCGGATCC ATG AAA ATG GTG ATA CCA GGC-3´) and ORF-R (5´-CCCAAGCTT ATA GAC AAG AGC AAA CCT AGC3´), the most coding region of pxCCE016b cDNA was obtained. The PCR product was retrieved and purified, and then inserted into the pMD18-T vector to construct the pMD18-pxCCE016b plasmid. After digested with restriction endonucleases BamHI and HindIII enzymes, the target fragment was ligated with T4 DNA ligase into the expression vector pET-32a(+) (Tiangen, China), which was also digested by the same restriction endonucleases enzymes before ligation. The construction diagram sees Fig. 8. The constructed plasmid solution was transformed into BL21 (DE3) competent cells (TaKaRa, Japan). After the positive colonies were selected and identified by restriction endonucleases BamHI and HindIII enzymes, the accuracy of the expressed plasmid was also verified by sequencing again. Subsequently, the BL21 (DE3) competent cells containing
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+
HindIII (173)
pET-pxCCE016b pxCCE016b
National Natural Science Foundation of China (31501664), the President Foundation of Guangdong Academy of Agricultural Sciences, China (201514), the Science and Technology Planning Project of Guangdong Province, China (2013B050800019 and 2014B070706017) and the Agro-Scientific Research Special Fund in the Public Interest, China (201103021).
References
T7 promoter
BamHI (198)
Fig. 8 Construction diagram of the pET-pxCCE016b.
recombinant plasmid was cultivated in Lucia-Bertani (LB) liquid medium and added the isopropl-D-thiog-alactoside (IPTG) to terminal concentration of 0.6 mmol L–1. Harvested the bacteria cells after 4–6 h cultivation with shaking and confirmed the expected fusion protein by SDS-PAGE eletrophoresis.
5.8. Western blot analysis Once it has been confirmed that the expected protein could be expressed in the BL21 (DE3) cells, Western blot analysis was applied to further identify of expression. The BL21 (DE3) cells contained the pET-pxCCE016b plasmid was induced under the same condition as prokaryotic expression experiment. The expressed bacteria were collected by centrifugation and suspension again with phosphate buffered saline (PBS). After added with 5×SDS-PAGE loading buffer (contains β-mercaptoethanol, Tiangen, China), the harvested bacteria cells were denatured at 100°C for 8–10 min and 10 μL of products per land was loaded on SDS-PAGE gel for electrophoresis analysis. Subsequently, the expression products were transferred from SDS-PAGE gel to a PVDF membrane by the Trans-Blot SD Semi-Dry Electrophoretic Transfer cell (Bio-Rad, USA) at 20 V for 15 min. The following Western blot analysis was carried out according to the reported procedures (Feng et al. 2012). When the PVDF membrane was incubated, the anti His-Tag mouse monoclonal antibody and affinipure goat anti-mouse IgG (H+L) were used as the primary and secondary antibody respectively. Finally, the color reaction of the membrane was carried out with DAB Kit (Chaorui, China).
Acknowledgements This study was funded by following research programs: the
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