Cloning, phylogenetic research, and prokaryotic expression study of the metabolic detoxification gene EoGSTs1 in Empoasca onukii Matsuda

Preparation of insect samples

E. onukii Matsuda used in this study is a sensitive strain that has been cultured in the laboratory for many generations. Insects were maintained in a light incubator with a constant temperature of 25 °C ± 1 °C, a humidity of 70% ± 5%, and a photoperiod of 16:8 (L:D). Small green leafhoppers of approximately the same size were starved for 1 h and then treated with 2.5 µg/mL of thiamethoxam for 48 h. Individuals who survived the treatment were collected at 20 insects/tube, flash frozen in liquid nitrogen, and stored in a −80 °C freezer.

Extraction and examination of total RNA

Total RNA was extracted using the TRIzol^® PlusRNA Purification Kit (Cat. No. 12183-555, Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. The extracted RNAs were examined by NanoDrop quantification and electrophoresis. Three microliters of each extracted RNA sample were used in agarose gel (1%) electrophoresis.

Primer design and template synthesis

Primers were designed using Primer Premier 6.0 (Premier Biosoft International, Palo Alto, CA, USA). Specific primer sequences are listed in Table 1. The 5′ rapid amplification of cDNA ends (RACE) and 3′ RACE templates were synthesized using a GeneRacer™ Kit (Cat. No. 12183-555, Invitrogen, Carlsbad, CA, USA). The cDNA template used for intermediate fragment PCR was synthesized using the SuperScript™ III First-Strand Synthesis SuperMix (Cat. No. 18080-400, Invitrogen, Carlsbad, CA, USA) for qPCR Kit.

PCR: The reaction system and conditions for 5′ RACE PCR, intermediate fragment PCR, and 3′ RACE PCR are presented in Tables S1–S10.

Prokaryotic expression and purification

Full-length splicing primers were designed based on PCR-based accurate synthesis (PAS). Protective bases were designed at both ends of the primers to synthesize the EoGSTs1-TARGET gene, which connected the site between the Nde I and Xba I sites of vector PCZN1. The obtained recombinant plasmid PCZN1-EoGSTs1-TARGET was transferred into Escherichia coli strain Top10, and positive clones were selected for sequencing and restriction enzyme digestion to ensure the correct expression plasmid was obtained through subcloning.

Approximately one µL of the constructed expression plasmid was added to 100 µL of Arctic Express E. coli, placed on the ice for 20 min, heat-shocked for 90 s at 42 °C, placed on ice for 5 min, added 600 mL of LB culture medium, and placed on a shaker for 1 h at the frequency of 220 rpm and 37 °C. After centrifugation, all the samples were coated onto LB plates that contained 50 µg/mL Amp (ampicillin), and incubated overnight at 37 °C. Monoclones were selected and inoculated into three mL of LB medium containing 50 µL/mL, placed on a shaker overnight at the frequency of 220 rpm and 37 °C, inoculated into 30 mL of LB medium (containing 50 µL/mL AMP) at a ratio of 1:100 the next day, and placed on a shaker at a frequency of 220 rpm and 37 °C when the OD600 value of the culture reached 0.6–0.8. Then, one mL of the culture was collected and centrifuged for 2 min at 10,000 rpm at room temperature. The supernatant discarded, and the pellet was resuspended in 100 µL 1 × sample loading buffer. IPTG was added to the remaining culture until the final concentration was 0.5 mM and placed on a shaker overnight at a frequency of 220 rpm and 11 °C to induce the expression of the fusion protein. Then, one mL of the culture was collected and centrifuged for 2 min at 10,000 rpm at room temperature, the supernatant was discarded, and the pellet was resuspended in 100 µL of 1 × sample loading buffer. The remaining culture was centrifuged for 10 min at 4,000 rpm, the supernatant was discarded, and the pellet was resuspended in phosphate-buffered saline (PBS). The cells in suspension were then lysed by sonication, the supernatant and pellet were resuspended in sample loading buffer, detected by 12% SDS-PAGE, and stained with Coomassie brilliant blue.

The results of the preliminary experiment showed that the target protein was soluble. Therefore, the supernatant was purified by Ni affinity chromatography. The supernatant solution (flow rate: 0.5 mL/min) was loaded onto the Ni-IDA-Sepharose Cl-6B affinity chromatography column (pre-equilibrated with Ni-IDA binding buffer) with a low-pressure chromatography system and washed with Ni-IDA binding buffer at the flow rate of 0.5 mL/min until the OD280 value of the effluent reached the baseline. The column was washed with Ni-IDA washing buffer (20 mM Tris–HCl, 20 mM imidazole, 0.15 M NaCl, pH 8.0) at the flow rate of one mL/min until the OD280 value of the effluent reached the baseline. Then, the target protein was eluted with Ni-IDA elution buffer (20 mM Tris–HCl, 250 mM imidazole, 0.15 M NaCl, pH 8.0) at a flow rate of one mL/min, and effluent was collected. The above-mentioned protein solution was poured into a dialysis bag, and PBS (3.58 g Na₂HPO₄⋅12H₂O, 8.8 g NaCl, 0.2 g KCl, 0.27 g KH₂P0₄) was used for overnight dialysis, then detected by 12% SDS-PAGE.

Western blot analysis

A three-µL sample was loaded into a well of a polyacrylamide gel. After running the stacking gel at 90 V and then increased to 200 V until the end of electrophoresis, conducted PVDF transfer (constant pressure 100 V, 1.5 h, constant current 250 mA), the membrane was washed four times (5 min each time) with phosphate-buffered saline-Tween 20 (PBST) and then kept in the blocking buffer containing 5% non-fat milk for 1 h at 37 °C. The membrane was incubated overnight with a term primary antibody that was diluted in the blocking buffer (dilution rate, 1:1000). The next day, the membrane was washed four times (5 min each time) with PBST. The membrane was then incubated with a secondary HRP antibody that was diluted with the blocking buffer with 5% milk (dilution rate, 1:5000) for 1 h at 37 °C. Then, the membrane was washed four times (5 min each time) in a clean box, developed, and exposed by the ECL (electrochemiluminescence) method.

Enzyme activity determination

Parameters of enzymatic kinetic: The determination method was based on Li et al. (2018a); Li et al. (2018b) with minor modifications. The reaction system consisted of phosphate buffer (100 mmol/L), rEoGSTs1 protein (0.12 mg), and GSH (0.01, 0.02, 0.04, 0.06, 0.08, and 0.1 mmol/L). After mixing the above solution, the mixture was incubated with 1-chloro-2,4-dinitrobenzene (CDNB) solution (10 mmol/L) at 35 °C for 15 min, then 20 µL of the CDNB solution was added into the mixture, and the volume was set to 200 µL. Subsequently, the absorbance was measured at a wavelength of 340 nm in the Multiskan GO 1510 spectrophotometer (Thermo Fisher Scientific, Vantaa, Finland), 30 s for each time and for a total of 2 min. Enzyme activity was calculated using the recombinant-inactivating protein as control. The specific activity was calculated according to the following formula, and the obtained data were submitted to GraphPad Prism 8.0.1 to calculate for enzyme kinetics using the following equation: ${\displaystyle \text{Specific activity}\left(\mathrm{\mu }\mathrm{mol}∕min\cdot \mathrm{mg}\right)=\frac{\Delta \mathrm{OD}340\times \mathrm{V}}{\epsilon \times \mathrm{T}\times \mathrm{L}\times \mathrm{E}}.}$ Note: OD340: Change in absorbance per unit time; V: enzymatic reaction volume (200 mL); ε: extinction coefficient of product (9.6 mmol/cm); T: reaction time (2 min); L: optical path (one cm); and E: amount of enzyme added (0.12 mg).

To determine the effect of temperature on the activity of the purified rEoGSTs1 protein, Glutathione S-transferase (GSH-ST) assay kit (Colorimetric method) was used (Cat. No. A004, Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China), following the manufacturer’s protocol. Approximately 0.12 mg of the rEoGSTs1 protein was added into the reaction system and then was incubated at 5 °C, 15 °C, 25 °C, 35 °C, 45 °C, and 60 °C for 15 min.

To determine the effect of pH on the activity of the purified rEoGSTs1 protein, Glutathione S-transferase (GSH-ST) assay kit (Colorimetric method) (Cat. No. A004, Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China) was used, following the manufacturer’s protocol. The pH of the reaction system was adjusted with PBS to 5.0, 6.0, 7.0, 8.0, and 9.0.

Data processing

The EoGSTs1 full-length cDNA was assembled from the DNA fragments using Seqman 7.1.0 software (DNAStar, Madison, WI, USA). Homology analysis of EoGSTS1 protein sequences in the NCBI database was performed using BLAST (Johnson et al., 2008). The physical and chemical properties of the EoGSTS1 protein were analyzed using software tools provided in the ExPASy-ProParam website (http://www.expasy.org), and cluster analysis was performed using MEGA 6.0 (Tamura et al., 2013). Clustal Omega (Sievers et al., 2011) was used in amino acid sequence alignment of EoGSTS1 and the GSTs of four other insects of order Hemiptera. The secondary structure of EoGSTS1 was predicted by CLC Genomics Workbench 11.0.1 (CLC Bio, Aarhus, Denmark), whereas the three-dimensional (3D) structure was predicted using I-TASSER software (Roy, Kucukural & Zhang, 2010). The functional domains were predicted based on the amino acid sequence deduced from the nucleotide sequence of the EoGSTs1 gene by searching the conserved domain database (CDD) in the NCBI database (Marchler-Bauer et al., 2006). Phylogenetic reconstruction of the GSTs was performed using the maximum likelihood (ML) method and Bayesian inference (BI) with the assistance of iqtree 1.6.1 (Nguyen et al., 2015) and MrBayes 3.2 (Ronquist et al., 2012).

Detection results of total RNA

Both the 28S RNA and 18S RNA bands were observed as distinct bands (Fig. 1), suggesting that the extracted RNAs were of good quality. The OD260/280 ratio of the RNA was 1.92, indicating that the collected RNA was of high purity with no degradation.

Amplification of the full-length cDNA

The 5′ end of the EoGSTs1 cDNA fragment obtained by 5′ RACE was 223 bp in length. The middle fragment of the EoGSTs1 amplified by intermediate fragment PCR was 480 bp in size. The 3′ end of the EoGSTs1 cDNA obtained by 3′ RACE was 431 bp in length (Figs. 2A–2C). The full-length cDNA of EoGSTs1 was an 841-bp sequence obtained through sequence assembly of the three DNA fragments; its coding region is 624-bp long, encoding a total of 207 amino acids, including the start codon ATG and the stop codon TGA (Dataset S1). The noncoding region at the 5′ end is 99 bp in length, and the 3′ end noncoding region is 118 bp in length followed by a 16-bp poly(A) tail (Fig. 3).

Taxonomic identification of EoGST

The amino acid sequence of EoGST is highly homologous to GSTs in Sub psaltriayangi (AVC68800.1), Locusta migratoria (AHC08043.1), Daktulosphaira vitifoliae (AUN35388.1), and Leptinotarsa decemlineata (APX61045.1), with a similarity of 53%, 52%, 52%, and 50%, respectively.

The amino acid sequence of EoGST obtained in small green leafhoppers is shown in Table 2 along with 54 other GST sequences belonging to six GST families. Cluster analysis using the adjacency method demonstrated that the EoGST gene is clustered with the Sigma subfamily of GSTs (a high bootstrap value of 99%), indicating that EoGST is closely related to this particular subfamily (Fig. 4). However, it did not cluster with GSTs in the Delta or Epsilon subfamilies, indicating that it is distantly related to these two subfamilies. Based on these findings and mammalian GST gene nomenclature, the nucleotide sequence of the E. onukii GST (EoGST, cluster-166.0) was initially named as EoGSTs1, and the encoded protein sequence was designated as EoGSTS1.

Bioinformatics analysis

Physical and chemical properties of the EoGSTS1 protein

The molecular weight of EoGSTS1 is 23.68932 kDa, with a calculated isoelectric point (IEP) of 6.00. The number of negatively and positively charged residues is 30 and 29, respectively. Its N-terminus begins with methionine, and the half-life of the protein is 30 h. The instability index of the protein in solution is 31.87, the fat coefficient is 78.26, and the total average hydrophobicity index is −0.376. The protein contains a total of 3,331 atoms, including 1,078 carbon atoms, 1,662 hydrogen atoms, 276 nitrogen atoms, 305 oxygen atoms, and 10 sulfur atoms. It consists of 20 different types of amino acids, among which the top three are alanine (Ala, A), lysine (Lys, K), and glutamic acid (Glu, E), with respective counts of 19 (9.2%), 19 (9.2%), and 16 (7.7%). The rarely occurring amino acids included Cys (C) and His (H), with counts of 2 (1%) and 1 (0.5%), respectively (Table 3).

Table 3:

The chemical composition of the cloned fragment of EoGSTs1.

Amino acid	Number	Proportion of quantity	Mass ratio	Amino acid	Number	Proportion of quantity	Mass ratio
Ala (A)	19	9.20%	6.18%	Lys (K)	19	9.20%	10.14%
Arg (R)	10	4.80%	6.36%	Met (M)	8	3.90%	4.36%
Asn (N)	6	2.90%	2.89%	Phe (F)	9	4.30%	5.43%
Asp (D)	14	6.80%	6.80%	Pro (P)	12	5.80%	5.04%
Cys (C)	2	1.00%	0.88%	Ser (S)	7	3.40%	2.68%
Gln (Q)	6	2.90%	3.20%	Thr (T)	8	3.90%	3.48%
Glu (E)	16	7.70%	8.59%	Trp (W)	6	2.90%	4.47%
Gly (G)	14	6.80%	3.84%	Tyr (Y)	10	4.80%	6.61%
His (H)	1	0.50%	0.57%	Val (V)	13	6.30%	5.56%
Ile (I)	13	6.30%	6.22%	Pyl (O)	0	0.00%	0.00%
Leu (L)	14	6.80%	6.70%	Sec (U)	0	0.00%	0.00%

DOI: 10.7717/peerj.7641/table-3

EoGSTS1 protein sequence alignment

The catalytic domain of the protein contains 69 amino acid residues, as revealed in the SMART software analysis (Letunic, Doerks & Bork, 2015). Comparison of the protein sequences of EoGSTS1 and the GSTs of four other insects of order Hemiptera using Clustal Omega (Fig. 5) revealed the existence of multiple conserved domains.

Analysis of the EoGSTS1 protein structure

Based on its protein sequence, neither signal peptide nor transmembrane helix domains are present in the protein, but four potentially linear antigenic epitopes may exist in the protein sequence (amino acids 31–51, 81–88, 112–123, and 169–180).

The EoGSTS1 protein contains nine α helices, two β sheets with a protein kinase C phosphorylation site, and one N-glycosylation site (Fig. 6). The protein belongs to the superfamily of GSTs and shares sequence similarities with GSTs in other insects (Fig. 7).

The protein sequence of EoGSTS1 contains two Asn-X-Ser/Thr motifs, one of which is an N-glycosylation site. The protein sequence also contains eight other potential phosphorylation sites: four serine phosphorylation sites located at positions 95, 118, 152, and 166 in the peptide chain; two threonine phosphorylation sites located at positions 47 and 125 in the peptide chain; and two tyrosine phosphorylation sites located at positions 148 and 188 in the peptide chain (Table 4).

Table 4:

Predicted phosphorylation sites.

Position	Sequence	Score	Classification	Position	Sequence	Score	Classification
95	DEIVSVVDE	0.986	*S*	47	IKPNTPWGK	0.801	*T*
118	ERKASLKEP	0.998	*S*	125	EPVLTQTVP	0.597	*T*
152	NGKFSWADV	0.985	*S*	145	ENKGYLANG	0.932	*Y*
166	SDHMSNMNG	0.546	*S*	188	RERVYAIPK	0.890	*Y*

DOI: 10.7717/peerj.7641/table-4

The 3D structure of the EoGSTS1 protein was predicted using the Blattella germanica GST (BgGST) protein (PDB ID: 4Q5R) as reference (as shown in Fig. 8, the C-terminus is denoted in red and the N-terminus in blue). The C-terminus is composed of 207 amino acids, and the N-terminus contains 204 amino acids. The C-score of the protein structural comparison is 1.21, indicating that it is highly similar to 4Q5R in terms of protein folding and secondary structure. The TM-score, which represents the structural similarity between the target sequence (EoGSTS1) and the template protein sequence (BgGST), is 0.88 ± 0.07.

Phylogenetic analysis

The phylogenetic tree of the full-length EoGSTS1 protein sequence and GST sequences of insects from five other orders, including Hemiptera, Orthoptera, Coleoptera, Odonata, and Hymenoptera (Table 5), were constructed using BI phylogenetic analysis and an ML-based method. The topological structures of the phylogenetic trees generated using either ML or BI displayed substantial similarities (Fig. 9). Cluster analysis indicated that the EoGSTS1 is clustered with known insect GSTs of order Hemiptera but not with GSTs from insects of order Odonata or Hymenoptera. These results suggest that E. onukii Matsuda is most closely related to insects of order Hemiptera and relatively closely related to those in order Orthoptera but distantly related to insects of orders Hymenoptera, Coleoptera, and Odonata.

Expression of the rEoGSTs1 protein

The recombinant expression plasmid pCzn1-EoGSTs1-target was transformed into E. coli. Arctic Express and the pCzn1/EoGSTs1 fusion protein was successfully expressed with the induction of IPTG. SDS-PAGE analysis showed that rEoGSTs1 was expressed in the supernatant and mainly occurred in a soluble form (Fig. 10A). Western blotting revealed a specific band in the corresponding position, which indicated that this band is the target band (Fig. 10B). The concentration of the target protein that was determined by the BCA method was 1.2 mg/mL.

Enzymatic analysis of EoGSTS1

The kinetic data of EoGSTS1 showed that the rEoGSTs1 protein could catalyze CDNB with Km of 0.21 ± 0.06 mmol/L and Vmax of 14.02 ± 1.40 µmol/min⋅mg (Fig. 11A). At different temperatures, the specific activity of EoGSTS1 initially gradually increased, and then decreased after reaching the highest value at 25 °C, and reached the lowest value at 60 °C. The lowest enzyme activity was about 4.13% of the highest one (Fig. 11B). At different pH levels, the enzyme activity of EoGSTS1 initially increased and then decreased with increasing pH, peaked at pH 7, and at pH 5, the enzyme activity was 73.67% of that at pH 7 (Fig. 11C).