Identification and characterisation of seventeen glutathione S-transferase genes from the cabbage white butterfly Pieris rapae

Accepted Manuscript Identification and characterisation of seventeen glutathione Stransferase genes from the cabbage white butterfly Pieris rapae

Su Liu, Yu-Xing Zhang, Wen-Long Wang, Bang-Xian Zhang, ShiGuang Li PII: DOI: Reference:

S0048-3575(17)30104-9 doi: 10.1016/j.pestbp.2017.09.001 YPEST 4111

To appear in:

Pesticide Biochemistry and Physiology

Received date: Revised date: Accepted date:

8 March 2017 30 August 2017 2 September 2017

Please cite this article as: Su Liu, Yu-Xing Zhang, Wen-Long Wang, Bang-Xian Zhang, Shi-Guang Li , Identification and characterisation of seventeen glutathione S-transferase genes from the cabbage white butterfly Pieris rapae. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Ypest(2017), doi: 10.1016/j.pestbp.2017.09.001

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ACCEPTED MANUSCRIPT Identification and characterisation of seventeen glutathione S-transferase genes from the cabbage white butterfly Pieris rapae Su Liu# , Yu-Xing Zhang# , Wen-Long Wang, Bang-Xian Zhang, Shi-Guang Li*

These authors contributed equally to this work.

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#

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College of Plant Protection, Anhui Agricultural University, Hefei, Anhui 230036, China

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This manuscript includes 27 pages, 6 figures, 3 tables, 5 supplementary files

Corresponding author:

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Shi-Guang Li, College of Plant Protection, Anhui Agricultural University, 130 West

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Changjiang Road, Hefei, Anhui 230036, China.

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E-mail: [email protected]

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Abstract Insect glutathione S-transferases (GSTs) play essential roles in the detoxification of insecticides and other xenobiotic compounds. The cabbage white butterfly, Pieris rapae, is an economically important agricultural pest. In this study, 17 cDNA sequences encoding

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putative GSTs were identified in P. rapae. All cDNAs include a complete open reading

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frame and were designated PrGSTd1–PrGSTz2. Based on phylogenetic analysis, PrGSTs

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were divided into six classes (delta, epsilon, omega, sigma, theta and zeta). The exon- intron organizations of these PrGSTs were also analysed. Recombinant proteins of eight PrGSTs

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(PrGSTD1, PrGSTD2, PrGSTE1, PrGSTE2, PrGSTO1, PrGSTS1, PrGSTT1 and PrGSTZ1)

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were heterologously expressed in Escherichia coli, and all of these proteins displayed glutathione-conjugating activity towards 1-chloro-2,4-dinitrobenzene (CDNB). Expression

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patterns in various larval tissues, at different life stages, and following exposure to sublethal

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doses of abamectin, chlorantraniliprole or lambda-cyhalothrin were determined by reverse transcription-quantitative PCR. The results showed that PrGSTe3, PrGSTs1, PrGSTs2, and

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PrGSTs4 were mainly transcribed in the fat body, while PrGSTe2 was expressed predominantly in the Malpighian tubules. Four genes (PrGSTe2, PrGSTo4, PrGSTs4 and PrGSTt1) were mainly expressed in fourth- instar larvae, while others were ubiquitously expressed in egg, larval, pupa and/or adult stages. Abamectin treatment significantly upregulated ten genes (PrGSTd1, PrGSTd3, PrGSTe1, PrGSTe2, PrGSTo1, PrGSTo3, PrGSTs1, PrGSTs3, PrGSTs4 and PrGSTt1). Chlorantraniliprole and lambda-cyhalothrin treatment significantly upregulated nine genes (PrGSTd1, PrGSTd2, PrGSTe1, PrGSTe2, PrGSTe3, PrGSTs1, PrGSTs3, PrGSTs4 and PrGSTz1) and ten genes (PrGSTd1, PrGSTd3,

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PrGSTe1, PrGSTe2, PrGSTo1, PrGSTo2, PrGSTs1, PrGSTs2, PrGSTs3 and PrGSTz2), respectively. These GSTs are potentially involved in the detoxification of insecticides.

Keywords

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Pieris rapae, GST, transcriptome analysis, insecticide treatment, detoxification

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1. Introduction Glutathione S-transferases (GSTs) are a superfamily of detoxifying enzymes present in both vertebrates and invertebrates [1]. In insects, GSTs play important roles in the detoxification of various harmful xenobiotic and endobiotic compounds, such as synthetic

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insecticides, plant allelochemicals, and lipid peroxides [2]. GSTs are able to catalyse the

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conjugation of reduced glutathione (GSH) with xenobiotics and endobiotics, making them

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less toxic and easier to excrete from cells [3]. Some other GSTs noncatalytically bind endogenous and exogenous compounds rather than metabolising them [4]. Apart from the

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detoxification function, insect GSTs are also required for odorant inactivation, ecdysteroid

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biosynthesis and larval development [5-7]. Furthermore, a number of GSTs have the peroxidase activity that protect against oxidative stress [8-9].

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There are two types of GSTs present in insects: cytosolic and microsomal [1]. Most

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insect GSTs belong to the cytosolic group, and they have been divided into six classes (delta, epsilon, omega, sigma, theta and zeta) according to their sequence identity, genomic

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structure, and biochemical properties [10]. GSTs that cannot be classified using this nomenclatural system are assigned to an 'unclassified' subgroup [10]. GSTs have two domains that are responsible for the detoxification function: a GSH binding domain (G-site), which is conserved in the N-terminal region of different classes of GSTs, and a hydrophobic substrate binding domain (H-site), which is more variable and located in the C-terminal region [3]. Upregulation of GST genes and enhanced activity of GST proteins has been associated with insecticide detoxification in many insect species [4]. Also, heterologously expressed

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insect GST proteins are capable of metabolising a variety of insecticides including organochlorines, organophosphates, and pyrethroids [3, 11-12]. Recently, RNA interference (RNAi) has been used to investigate the function of insect GSTs, and in many species including Aedes aegypti, Locusta migratoria, Nilaparvata lugens and Bemisia tabaci, an

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involvement in detoxifying insecticides has been demonstrated [13-16].

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To date, 37, 28, 8, 36, 28, 29, 24, 9, 28 and 14 cytosolic GST genes have been identified

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in the model insect species Drosophila melanogaster, Anopheles gambiae, Apis mellifera, Tribolium castaneum, Dendroctonus ponderosae, Leptinotarsa decemlineata, Acyrthosiphon

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pisum, N. lugens, L. migratoria and Rhodnius prolixus, respectively [15, 17-20]. GST genes

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have also been discovered in the model species Bombyx mori (23 genes) and Plutella xylostella (22 genes) of the Order Lepidoptera, and in the agricultural pests Spodoptera

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litura (37 genes) and Cnaphalocrocis medinalis (25 genes) [21-24]. However, these species

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are all moths, and GST genes in butterflies remain poorly characterised. The small white butterfly, Pieris rapae, is a severe agricultural pest distributed

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worldwide that causes serious yield losses in Brassicaceae crops [25]. In the past decades, management of P. rapae in China mainly relies on the spraying of insecticides. Abamectin, chlorantraniliprole and lambda-cyhalothrin are three insecticides widely used for controlling the pest insect [26]. Recently, however, these insecticides became less efficient to control the pest even at relatively high doses (S. Liu and S.-G. Li, personal observation). It is possible that GSTs in P. rapae contribute to the detoxification of insecticides. In this study, we identified 17 GST genes (PrGSTs) from P. rapae using previously released transcriptome datasets [27]. We also analysed the exon- intron organizations and

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phylogenetic relationships of these PrGSTs. Eight PrGST proteins were heterologously expressed in Escherichia coli, and their catalytic activities were investigated. Transcription of some genes varied in different larval tissues, at different developmental stages, and following exposure to various concentrations of abamectin, chlorantraniliprole and

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lambda-cyhalothrin. To our knowledge, this is the first report o n the large-scale identification

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and characterisation of GST genes in this economically important insect pest.

2. Materials and methods

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2.1. Insects

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P. rapae individuals used in this study originated from a colony collected from cabbage fields in an experimental farmland of Anhui Agricultural University, Hefei, Anhui, China.

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Larvae were reared on the leaves of Chinese cabbage (Brassica pekinensis) and adults were

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fed on a 10% (v/v) honey solution. Animals were reared at 25 ± 1°C with 65% relative humidity and a 16:8 h light:dark photoperiod.

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2.2. RNA extraction and cDNA synthesis Total RNA was isolated using RNAiso Plus reagent (Takara, Dalian, China) and treated with RNase-free DNase I (Takara, Dalian, China) to remove potential contaminants from genomic DNA. The quality and concentration of RNA were determined by agarose gel electrophoresis and NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE). First-strand cDNA was reverse-transcribed using the TransScript First-Strand cDNA Synthesis SuperMix (Transgen, Beijing, China).

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2.3. Homology searching and sequence verification cDNA sequences encoding GSTs were retrieved from previously released P. rapae transcriptome datasets [27] using the TBLASTN algorithm in the Basic Local Alignment Search Tool (BLAST) program [28]. Annotated GST protein sequences from model insect

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species (including D. melanogaster, A. gambiae, B. mori and P. xylostella) were used as

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queries with a cut-off E-value of 1 × 10-5 .

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To confirm that the identified GST sequences were not chimeric, gene-specific primers (Table S1) were designed to amplify complete or partial open reading frames (ORFs).

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First-strand cDNA from fourth- instar larvae was used as a template. PCR products were

Searching

for

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2.4. Bioinformatic analyses

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sequenced in both 5' and 3' directions.

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analysed by agarose gel, and DNA bands of the expected size were excised, purified, and

orthologs

was

performed

using

the

BLAST

program

residues

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(http://blast.ncbi.nlm.nih.gov/blast.cgi) with a cut-off E-value of 1 × 10-5 [28]. Catalytic were

predicted

by

searching

the

Conserved

Domain

database

(http://www.ncbi.nlm.nih.gov/structure/cdd/cdd.shtml) [29]. Multiple sequence alignment was performed using the Clustal Omega program (http://www.ebi.ac.uk/tools/msa/clustalo/) [30]. Phylogenetic tree construction was performed by MEGA6.0 software using the neighbour-joining method with the pairwise deletion option [31]. To evaluate the branch strength of the tree, a 1000 bootstrap replication analysis was performed [31]. The GenBank accession numbers of sequences used are listed in Table S2. The genomic DNA of P. rapae

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was download from a lepidopteran genome database (http://prodata.swmed.edu/LepDB/) [32], and the exon- intron structure of PrGSTs was determined by aligning cDNA sequences with

genomic

DNA

sequences

using

Splign

program

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(https://www.ncbi.nlm.nih.gov/sutils/splign/splign.cgi) [33].

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2.5. Protein expression and purification

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The complete ORFs of eight PrGSTs (PrGSTd1, PrGSTd2, PrGSTe1, PrGSTe2, PrGSTo1, PrGSTs1, PrGSTt1, and PrGSTz1) were cloned using gene-specific primers (Table

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S1) and ligated into the pEASY-Blunt E1 expression vector (Transgen, Beijing, China). Each

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recombinant protein will be expressed as a fusion protein with a N-terminal 6×His·tag. Each expression construct was verified by DNA sequencing, and the correct constructs were

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transformed into the E. coli OrigamiB (DE3) cells. Bacteria were cultured in Luria-Bertani

tetracycline)

and

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(LB) medium (containing 50 μg/ml of ampicillin, 30 μg/ml of kanamycin, and 12.5 μg/ml of grown

at

37

°C

until

OD600

was

0.6,

then

isopropyl

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β-D-1-thiogalactopyranoside (IPTG) was added to 0.5 mM final concentration. After growing at 30 °C for 6 h, cells were harvested by centrifugation at 3000 × g for 5 min, resuspended in lysis buffer [20 mM Tris·HCl (pH 7.4), 500 mM NaCl, 15% glycerol, and 1 mM phenylmethanesulfonyl fluoride (PMSF)], and lysed by sonication on ice using a probe sonicator (Xinzhi Biotech., Ningbo, China). The recombinant proteins were all in soluble form. Proteins were purified by using a HisTrap column (GE Healthcare, Uppsala, Sweden) with a linear gradient of 0–300 mM imidazole, and desalted by using a Centricon filter device (10 kD cut-off, Millipore, Ireland). The purity of recombinant protein was analyzed

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by 12% (w/v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The concentration of protein was measured using a Pierce BCA protein assay kit (Thermo Scientific, Wilmington, DE).

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2.6. Activity assay

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The 1-chloro-2,4-dinitrobenzene (CDNB) and GSH were purchased from Sigma-Aldrich

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(St Louis, MO) and dissolved in ethanol and H2 O, respectively. The activity assay was performed according to a protocol described by Samra et al [11]. Briefly, a 200 μl reaction

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mixture contained 200 ng protein, 1 mM CDNB, 5 mM GSH, and 1% (v : v) ethanol in 0.1

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M sodium phosphate buffer (pH 6.5). The increases of absorbance were monitored at 15 s intervals at 340 nm for 3 min. The CDNB-conjugating activity (μmol/min/mg) was

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calculated using the molar extinction coefficient (ε340 = 9600 M−1 cm−1 ) of the resultant

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2,4-dinitrophenyl- glutathione. The assays were biologically repeated three times, the absorbance was recorded on a Multiskan Go microplate reader (Thermo Scientific,

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Wilmington, DE).

2.7. Reverse transcription-quantitative PCR (RT-qPCR) RT-qPCR was used to investigate the expression profiles of PrGSTs in various larval tissues, including integument, fat body, midgut, and Malpighian tubules. Tissues were dissected from more than 100 fourth- instar larvae and stored at −80°C until RNA extractions were carried out. Transcription profiles were also investigated in different developmental stages, including 300 eggs, 60 fourth- instar larvae, 60 pupae, and a mixture of 30 adult males

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and 30 adult females. RNA isolation was performed as described in Section 2.2, and first-strand cDNA was synthesized using the ReverTra Ace qPCR RT kit (Toyobo, Osaka, Japan). Each cDNA sample was diluted to 10 ng/μl with nuclease-free water. Primers for RT-qPCR are listed in Table S1. Two housekeeping genes (18S rRNA and

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β-actin) were used as internal references to normalise target gene expression. In a pre-test,

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the amplification efficiency (listed in Table S1) of each primer pair was calculated from the

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slope of the log- linear portion of the curve generated by amplification from serially diluted cDNA samples. RT-qPCR was performed in triplicate in 96-well reaction plates (Bio-Rad,

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Hercules, CA). Each reaction (20 μl volume) contained 10 μl SYBR Green Real-time PCR

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Master Mix (Toyobo, Osaka, Japan), 0.4 μl (0.2 μM) of each primer, 1 μl (10 ng) cDNA template, and 8.2 μl nuclease-free water. RT-qPCR was performed on a CFX96 Real-Time

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System (Bio-Rad, Hercules, CA) with the following parameters: one cycle at 95°C for 30 s,

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and 40 cycles at 95°C for 5 s, and 60°C for 25 s. To confirm that only one single gene was detected by the fluorescence dye, a heat-dissociation protocol was added at the end of the

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thermal cycle. A no-template control and a no-reverse transcriptase control were included on each reaction plate to detect potential contamination. Reactions for all samples were independently repeated three times, and quantitative variation in gene expression among different samples was calculated by a modified version of the Pfaffl method [34].

2.8. Insecticide treatment Abamectin, chlorantraniliprole and lambda-cyhalothrin were purchased from Dr Ehrenstorfer GmbH (Augsburg, Germany), the purity of these insecticides are all ≥ 94%.

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The three chemicals were diluted with analytical- grade acetone to produce a stock solution from which serial decimal dilutions were carried out. In previous study, three sublethal doses, LD5 , LD20 and LD50 (5%, 20% and 50% of the test animals are killed at 24 h, respectively), for each insecticide were determined and the values are listed in Table S3.

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The freshly molted (<24 h) fourth- instar larvae were used for the bioassay. Larvae were

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placed into a clean glass petri dish (9-cm diameter) containing a piece (5 × 5 cm) of fresh

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cabbage (B. pekinensis) leaves. A droplet of 1 μl of insecticide solution at each dose was applied topically on the dorsal part of larval middle abdomen with a 10 μl microsyringe

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(Gaoge Industry and Trade Co. Ltd., Shanghai, China). Control insects were treated with 1 μl

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of acetone only. The rearing conditions for treated larvae were controlled at 25 ± 1°C and 65% relative humidity. At 6 h after the treatment, surviving insects were collected for

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RT-qPCR analysis (as described in Section 2.7). Each treatment was biologically repeated

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three times, and in each repeat 30 larvae were used.

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2.9. Statistical analysis

Data were analysed using Data Processing System (DPS) software v9.5 [35]. Student's t-test and one-way analysis of variance (ANOVA) with Tukey's post-hoc test were performed, respectively, for comparing difference between two samples and differences among multiple samples. The level of significance was set at p <0.05.

3. Results 3.1. Identification and classification of P. rapae GSTs

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A total of 17 cDNA sequences encoding putative GSTs were identified from P. rapae transcriptome data and designated PrGSTd1–PrGSTz2 (Table 1). In order to confirm that the assembled PrGSTs were not chimeric, PCR analysis was performed and the results showed that all PrGSTs were amplified from larval cDNA with gene-specific primers (data not

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shown). DNA sequencing subsequently confirmed that the sequences of amplified PrGSTs

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were identical to those retrieved from the transcriptome data. All PrGST cDNAs included a

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complete ORF, and the size of deduced PrGST proteins ranged from 203 to 287 amino acid residues (Table 1), sharing between 12% and 76% amino acid sequence identity (Table S4).

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A BLASTX search of the best hits showed that PrGSTs share relatively high sequence

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identity (56–95%) with their respective orthologs from other lepidopteran species (Table 1). The results of Conserved Domain revealed G-sites in the N-terminal regions in 10 of the

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PrGSTs but not in PrGSTE1, PrGSTE2, PrGSTO1, PrGSTO2, PrGSTO3, PrGSTO4 and

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PrGSTZ2, and H-sites in the C-terminal region of all PrGSTs (Fig. 1). Phylogenetic analysis was performed to better understand the classification of PrGSTs.

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The 17 PrGSTs clearly segregated into six classes (delta, epsilon, omega, sigma, theta and zeta) (Fig. 2). Based on this classification, P. rapae possesses three delta-class GSTs, three epsilon GSTs, four omega GSTs, four sigma GSTs, one theta GST, and two zeta GSTs (Fig. 2; Table 1).

3.2. Exon-intron organization of PrGSTs The position of exons and introns in each PrGST gene was analysed by aligning a cDNA sequence with a genomic DNA sequence (Fig. 3). The size of one intron in PrGSTe1 gene

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was not identified due to gaps in the genomic DNA (Fig. 3). A total of 64 introns were found in the 17 PrGST genes. No intronless gene was observed. The donor- and acceptor-sites of these introns all conformed to the classical GT–AG splice junction rules (data not shown). Most PrGST genes have three or four introns, whereas PrGSTd1 and PrGSTo3 both have

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five introns (Fig. 3). The PrGSTd3 and PrGSTe2 possessed the smallest (two) and the largest

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(six) numbers of introns, respectively (Fig. 3).

3.3. Comparison of GSTs in P. rapae and other insects

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The number of annotated GSTs in P. rapae and other representative insect species

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belonging to different Orders (Lepidoptera, Diptera, Coleoptera, Hymenoptera, Hemiptera and Orthoptera) are listed in Table 2. This number clearly varies greatly between species,

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with a particularly large number (27 to 39 genes) in Diptera, Coleoptera and Orthoptera, but

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far fewer in the hymenopteran A. mellifera (10 genes) and the hemipteran N. lugens (11 genes) (Table 2). Within the Order Lepidoptera, P. rapae (17 genes) possesses fewer GSTs

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than P. xylostella (22 genes), B. mori (24 genes), C. medinalis (25 genes) and S. litura (37 genes) (Table 2). There are fewer delta (three) and epsilon (three) class GSTs in P. rapae than in other lepidopteran species (Table 2).

3.4. Functional characterisation of recombinant PrGST proteins Recombinant proteins of eight PrGSTs (PrGSTD1, PrGSTD2, PrGSTE1, PrGSTE2, PrGSTO1, PrGSTS1, PrGSTT1, and PrGSTZ1) were heterologously expressed in E. coli as histidine- fusion proteins (Fig. S1). The CDNB-conjugating activities of these proteins were

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characterised in vitro (Table 3). The results showed that one delta-class GST, PrGSTD2, had the highest activity (4.61 μmol/min/mg) towards CDNB than other PrGSTs; whereas a theta-class GST, PrGSTT1, exhibited the lowest activity (0.028 μmol/min/mg) to catalyse the conjugation of CDNB. The activities of other PrGSTs ranged from 0.49 to 1.57

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μmol/min/mg (Table 3).

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3.5. Expression of PrGSTs in different larval tissues and at different developmental stages Using RT-qPCR, expression profiles of PrGSTs were investigated in four different larval

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tissues (integument, fat body, midgut and Malpighian tubules). The results showed that four

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genes (PrGSTe3, PrGSTs1, PrGSTs2 and PrGSTs4) were mainly transcribed in the fat body; only one gene, PrGSTe2, was mainly expressed in Malpighian tubules (Fig. 4). None of the

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genes were specifically distributed in the integument or midgut (Fig. 4). The remaining

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genes were ubiquitously expressed in all the tested tissues or in at least two tissues. For example, PrGSTd1 was expressed predominantly in both the fat body and midgut, whilst

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PrGSTo2 was omnipresently transcribed in all the four tissues examined (Fig. 4). Expression patterns of PrGSTs at different developmental stages were also determined, and four genes (PrGSTe2, PrGSTo4, PrGSTs4 and PrGSTt1) were mainly expressed during the fourth- instar larval stage (Fig. 5). However, no genes were predominantly expressed in egg, pupal or adult stages. Some genes were mainly expressed in two or three developmental stages, such as PrGSTd1, which was predominantly expressed in larval, pupal and adult stages, while PrGSTe1 was transcribed mainly in larval and pupal stages (Fig. 5).

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3.6. Expression of PrGSTs in larvae exposed to insecticides To investigate whether the expression of PrGSTs respond to the synthetic insecticides abamectin, chlorantraniliprole and lambda-cyhalothrin, PrGST transcript levels were determined in larvae following exposure to sublethal doses (LD5 , LD20 , and LD50 ) of these

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insecticides. The result revealed that the expression of eight genes (PrGSTd1, PrGSTd3,

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PrGSTe1, PrGSTe2, PrGSTo1, PrGSTo3, PrGSTs1, PrGSTs3 and PrGSTs4) was

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significantly upregulated by LD50 dose of abamectin, compared with control insects (Fig. 6A). The LD5 and LD20 doses of abamectin also significantly elevated five (PrGSTe1,

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PrGSTe2, PrGSTs1, PrGSTs3 and PrGSTs4) and eight (PrGSTd3, PrGSTe1, PrGSTe2,

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PrGSTo3, PrGSTs1, PrGSTs3, PrGSTs4 and PrGSTt1) genes, respectively (Fig. 6A). Similarly, PrGST genes were significantly upregulated by chlorantraniliprole and

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lambda-cyhalothrin; specifically, chlorantraniliprole significantly stimulated PrGSTd1,

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PrGSTd2, PrGSTe1, PrGSTe2, PrGSTe3, PrGSTs1, PrGSTs3, PrGSTs4 and PrGSTz1 (Fig. 6B), whilst lambda-cyhalothrin significantly upregulated PrGSTd1, PrGSTd3, PrGSTe1,

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PrGSTe2, PrGSTo1, PrGSTo2, PrGSTs1, PrGSTs2, PrGSTs3 and PrGSTz2 (Fig. 6C). Conversely, three genes (PrGSTd2, PrGSTo4 and PrGSTz1) were significantly downregulated after exposure to different concentrations of abamectin (Fig. 6A). Similarly, chlorantraniliprole downregulated PrGSTd3, PrGSTo1, PrGSTs2, PrGSTt1 and PrGSTz2, while lambda-cyhalothrin decreased the mRNA levels of PrGSTe3, PrGSTs4, PrGSTt1 and PrGSTz1 (Fig. 6B, C).

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4. Discussion Since whole-genome information for P. rapae is still unavailable, searching of transcriptome datasets can be used to identify specific genes, including GST genes. This approach has been used successfully for other insects lacking genomic data, such as S. litura

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[21], Laodelphax striatellus [36], C. medinalis [22], and Bactrocera dorsalis [37]. In the

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present study, we identified 17 PrGST genes, which is greater than the number present in A.

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mellifera [38], L. striatellus [36], and N. lugens [15], but less than the number in some other insects, especially lepidopteran species. For example, there are 22, 24, 25 and 37 GST genes

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in P. xylostella, B. mori, C. medinalis and S. litura, respectively [21-24]. Although we

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performed exhaustive searches of the available transcriptome data, it is of course possible that additional GST genes may be present in P. rapae that were not detected in this study.

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Based on phylogenetic analysis, the 17 PrGSTs were divided into six classes (delta,

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epsilon, omega, sigma, theta and zeta) (Fig. 2). Delta and epsilon are insect-specific classes, and GSTs from these two classes often have detoxification functions and have been related to

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resistance to insecticides [3-4]. For instance, delta GSTs from Culex pipiens and Cydia pomonella are able to metabolise 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) and lambda-cyhalothrin, respectively [11-12]. Furthermore, silencing of epsilon GSTs in Aedes aegypti and B. dorsalis by RNAi increases susceptibility to pyrethroid and malathion, respectively [13, 39]. P. rapae has fewer delta and epsilon GSTs compared with other lepidopteran species such as P. xylostella, B. mori, C. medinalis and S. litura. This is probably due to within-species variation, and the deficit of delta and epsilon GSTs may be functionally compensated by GSTs from other classes.

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To date, GSTs from many different insect species have been heterologously expressed and purified [8-9, 11-12]. Furthermore, the biochemical characteristics of these proteins have been analysed in vitro, and the results showed that the catalytic properties of insect GSTs varied greatly. For instance, an omega GST (AccGSTO1) from Apis cerana cerana

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displayed low activity for CDNB (0.015 μmol/min/mg) [40], whilst a delta GST (CpGSTD1)

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from Culex pipiens had strong ability to conjugate CDNB (> 40 μmol/min/mg) [11]. In the

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current study, recombinant proteins of eight PrGSTs were produced in the E. coli expression system and all of them displayed catalytic activities towards CDNB in the presence of GSH

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(Table 3). The result indicated that these PrGSTs were functional proteins and might play a

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significant role in xenobiotic detoxification. Although biochemical study was not performed for other PrGSTs, they were expected to have catalytic functions and might be involved in

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important physiological pathways.

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The insect fat body, midgut, and Malpighian tubules are well-known for their critical roles in the detoxification of xenobiotic compounds [41-43]. In many lepidopteran species

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such as B. mori, P. xylostella and C. medinalis, several GST genes are highly expressed in these tissues [22-24]. In this study, we found four genes (PrGSTe3, PrGSTs1, PrGSTs2, and PrGSTs4) that were expressed predominantly in the fat body, and one gene (PrGSTe2) that was mainly transcribed in Malpighian tubules (Fig. 4). These PrGSTs are likely associated with the detoxification of xenobiotics. Furthermore, we found several genes that were at a moderate expression level in integument (Fig. 4). Transcription of GST genes in integument has been reported in many insect species including A. cerana cerana, S. litura, and Bombus ignitus [8, 44-45], implying a possible role of GSTs in the initial detoxification of xenobiotic

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compounds. Other PrGST genes were ubiquitously expressed in various tissues, indicating a basic but presumably important physiological function in P. rapae. In many insect species, GST mRNA levels vary greatly during different life stages. For instance, GSTs1 in P. xylostella is highly expressed in egg and larval stages, but not in pupal

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and adult stages [24]. By contrast, a sigma-class GST gene in Chilo suppressalis is expressed

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most highly in adults [46]. In the present study, four PrGSTs (PrGSTe2, PrGSTo4, PrGSTs4

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and PrGSTt1) were predominantly expressed during the fourth- instar larval stage (Fig. 5), suggesting that they might be related to the detoxification of secondary metabolites from

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host plants and insecticides. Several GST genes in N. lugens, L. striatellus, S. furcifera, P.

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xylostella and B. dorsalis are enriched during the egg stage and might regulate hormones throughout embryonic development [15, 24, 36-37]. However, no egg stage-specific GSTs

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were identified in P. rapae.

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Abamectin, chlorantraniliprole and lambda-cyhalothrin have been widely used to control a variety of insect pests, including P. rapae [26]. Recently, variation in susceptibility to these

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insecticides was observed in N. lugens [9], P. xylostella [47-48], S. exigua [49], C. medinalis [50], C. suppressalis [51-52] and Apolygus lucorum [53]. Although mutations in some key proteins such as the glutamate-gated chloride channel, ryanodine receptor, and voltage- gated sodium channel can contribute to reduced susceptibility [53-55], detoxifying enzymes such as GSTs, esterases and cytochrome P450s may also play important roles in the detoxification insecticides [12, 56-59]. Additionally, insect GSTs may also be involved in protecting against the oxidative stress induced by insecticides [8-9]. In this study, ten, eight, and ten PrGSTs were upregulated following exposure to chlorantraniliprole, abamectin, and

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lambda-cyhalothrin, respectively (Fig. 6). These genes are therefore potential candidates involved in the detoxification of the tested insecticides. Downregulation of PrGSTs was also observed in larvae treated with insecticides (Fig. 6). Decreased GST mRNA levels following insecticide exposure has also been reported for

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many other insect species such as N. lugens, L. striatellus, S. furcifera, B. dorsalis, C.

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medinalis and L. decemlineata [15, 20, 22, 36-37]. Han et al [20] suggested that

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downregulation of a subset of GSTs may be an adaptive mechanism in insects that reduces the total activity of GST enzymes to prevent excessive GST activity from exhausting the

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supply of GSH. However, functional studies are needed to test this hypothesis.

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In conclusion, we identified 17 GST genes in P. rapae by analysing previously published transcriptome data. The exon-intron organizations and phylogenetic relationships of PrGSTs

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were investigated, and the catalytic activities of eight PrGST proteins were also examined.

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PrGST genes displayed distinct expression profiles in various larval tissues and during different life stages. The transcription of several genes was induced following exposure to

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abamectin, chlorantraniliprole and lambda-cyhalothrin, consistent with an involvement in the detoxification of insecticides. This is the first report on the large-scale identification and characterisation of GST genes in P. rapae. The results will pave the way for a better understanding of detoxification enzymes in this insect species.

Conflict of interest The authors declare that there is no conflict of interest in this paper.

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Acknowledgements This work was supported by the National Key Research and Development Program of China (grant number 2016YFD0200205-7), the National Natural Science Foundation of China (grant number 31401734), and the Anhui Provincial Natural Science Foundation

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(grant number 1708085QC50).

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Figure captions Fig. 1. Predicted GSH binding sites (G-sites) and substrate binding sites (H-sites) of deduced PrapGST proteins.

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Fig. 2. Phylogenetic analysis of GSTs from Acyrthosiphon pisum (Ap-prefix), Anopheles

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gambiae (Ag), Apis mellifera (Am), Bactrocera dorsalis (Bd), Bombyx mori (Bm),

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Cnaphalocrocis medinalis (Cm), Drosophila melanogaster (Dm), Leptinotarsa decemlineata (Ld), Locusta migratoria (Lm), Nilaparvata lugens (Nl), Plutella xylostella (Px), Pieris

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rapae (Pr), Spodoptera litura (Sl), and Tribolium castaneum (Tc). Bootstrap support values

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based on 1000 replicates are indicated by colouring from green (0) to red (100) on each node. Insect GSTs are classified into six classes (delta, epsilon, omega, sigma, theta and

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zeta) and an 'unclassified' subgroup. The seventeen P. rapae GSTs (PrGSTs) are coloured

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red. GenBank accession numbers of sequences used are listed in Table S2.

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Fig. 3. Schematic diagram of the exon-intron structure of each PrGST gene.

Fig. 4. Relative expression levels of PrGSTs in various larval tissues. IG, integument; FB, fat body; MG, midgut; MT, Malpighian tubes. Expression levels in different tissues were normalized relative to that in the integument. Different lowercase letters indicate significant variation in transcription among different samples (one-way ANOVA with Tukey's post-hoc test, p < 0.05).

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Fig. 5. Relative expression levels of PrGSTs during different life stages. E, egg stage; L, fourth- instar larval stage; P, pupal stage; A, adult stage. Expression levels at different life stages were normalized relative to that in the egg stage. Different lowercase letters indicate significant variation in transcription among different samples (one-way ANOVA with

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Tukey's post-hoc test, p < 0.05).

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Fig. 6. Relative expression levels of PrGSTs in larvae exposed to three different concentrations (LD5 , LD20 , and LD50 ) of (A) abamectin, (B) chlorantraniliprole, and (C)

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lambda-cyhalothrin. The transcriptional level of each gene in insecticide-treated individuals

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was normalized relative to that in acetone-treated (control) individuals. * and ** indicate

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significant upregulation and downregulation, respectively (Student's t-test, p < 0.05).

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Table 1 Details of the 17 GSTs identified in P. rapae. Group Gene name

GenBank ORF

Mw

acc. no.

(kDa)

(aa)

pI

BLASTX top hit Identity Reference organism

Acc. no.

E-value

PrGSTd1KX229707 244

6.2

27.4 Amyelois transitella

XP_0131923911E-128 71

PrGSTd2KX229708 216

6.2

24.4 Danaus plexippus

EHJ63706

PrGSTd3KX229723 214

6.6

24.3 Papilio polytes

NP_0012983812E-95

Epsilon PrGSTe1 KX229709 225

8.7

26.3 Papilio xuthus

KPJ03136

2E-109 68

PrGSTe2 KX229710 253

6.3

28.7

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(%) Delta

AIZ46901

6E-109 61

P46430

2E-93

63

6.1

24.6 Manduca sexta

Omega PrGSTo1 KX229712 254

6.1

28.9 Danaus plexippus

EHJ65985

3E-137 72

PrGSTo2 KX229713 287

8.8

33.3 Bombyx mori

ABD36306

2E-110 56

PrGSTo3 KX229714 241

7.1

28.8 Chilo suppressalis

AKS40347

1E-145 81

PrGSTo4 KX229715 275

8.2

31.7 Danaus plexippus

EHJ65985

6E-105 57

Sigma PrGSTs1 KX229716 204

5.8

23.2 Amyelois transitella

XP_0131955713E-101 68

PrGSTs2 KX229717 205

6.8

23.4 Bombyx mori

NP_0010369943E-86

PrGSTs3 KX229718 203

9.0

23.6

AAF23078

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PrGSTe3 KX229711 215

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medinalis

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Cnaphalocrocis

9E-131 89

60

61

PrGSTs4 KX229719 206

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Choristoneura 4E-103 70

fumiferana

8.5

23.6 Papilio machaon

XP_0143644454E-81

59

7.0

26.5 Danaus plexippus

EHJ70012

5E-106 65

PrGSTt1 KX229720 224

Zeta

PrGSTz1 KX229721 219

7.1

25.1 Chilo suppressalis

AKS40351

2E-148 95

PrGSTz2 KX229722 219

9.4

25.3 Spodoptera litura

AIH07599

4E-99

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Theta

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Table 2 Comparison of GST genes in various insect species. Species

Delta Epsilon Omega Sigma Theta Zeta Unclassified Microsomal Total

Lepidoptera Pieris rapae 3 Plutella xylostella 5

2 2

0 2

– –

17 22

8

4

2

1

2

2

1

24

Cnaphalocrocis medinalis

4

9

3

5

0

2

2

–

25

Spodoptera litura 4 Drosophila 11

15 14

3 5

6 1

1 4

2 2

1 0

5 1

37 38

melanogaster Anopheles

12

8

1

1

2

1

3

3

31

gambiae Aedes aegypti

8

8

1

1

4

1

3

1

27

Culex

17

10

1

2

6

0

3

0

39

quinquefasciatus Tribolium

3

19

4

7

1

1

0

1

36

castaneum Dendroctonus ponderosae

6

12

2

2

1

0

–

28

Leptinotarsa

3

10

1

0

5

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4

5

4

4

1

2

1

30

1

4

1

1

0

2

10

5

0

2

8

3

1

0

0

19

vitripennis Acyrthosiphon

10

0

0

6

2

0

0

2

20

1

1

3

1

1

0

2

11

0

3

12

2

1

0

0

28

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Nasonia

pisum Nilaparvata lugens 2 Locusta migratoria

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Orthoptera

1 1

Bombyx mori

decemlineata Hymenoptera Apis mellifera

Hemiptera

4 2

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Coleoptera

4 5

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Diptera

3 5

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Order

10

Data are collated from [15, 17, 18, 20-24]. "–" denotes data are not shown in the literature (P. xylostella, C. medinalis and D. ponderosae), or that transcriptome searching was not performed (P. rapae).

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Table 3 The CDNB-conjugating activities of eight recombinant PrGST proteins. Activity (μmol/min/mg)

PrGSTD1 PrGSTD2

0.76 ± 0.08 4.61 ± 0.35

PrGSTE1 PrGSTE2

0.92 ± 0.05 1.57 ± 0.2

PrGSTO1 PrGSTS1

0.49 ± 0.07 0.93 ± 0.09

PrGSTT1

0.028 ± 0.006

PrGSTZ1

1.2 ± 0.15

AC C

EP T

ED

MA

NU

SC

RI

PT

Protein

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AC C

EP T

ED

MA

NU

SC

RI

PT

Graphical abstract

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Highlights A total of 17 GST genes (PrGSTs) were identified in Pieris rapae



Eight PrGST proteins were functionally characterised



Some PrGSTs display tissue- and developmental stage-specific expression



Several PrGST genes are significantly upregulated by insecticides



PrGSTs are potentially involved in insecticide detoxification

AC C

EP T

ED

MA

NU

SC

RI

PT

