LlU Zhen-gang, WANG Huan-huan, XUE Chao-bin

Key Laboratory of Pesticide Toxicology and Application Technique, College of Plant Protection, Shandong Agricultural University,Tai’an 271018, P.R.China

Abstract Laccase (EC 1.10.3.2) is known to oxidize various aromatic and nonaromatic compounds via a radical-catalyzed reaction,which generally includes two types of laccase, Lac1 and Lac2. Lac1 oxidizes toxic compounds in the diet, and Lac2 is known to play an important role in melanizing the insect exoskeleton. In this study, we cloned and sequenced the cDNA of the diamondback moth, Plutella xylostella Lac2 (PxLac2), from the third instar larvae using polymerase chain reaction (PCR)and rapid amplification of cDNA ends techniques. The results showed that the full-length PxLac2 cDNA was 1 944 bp long and had an open reading frame of 1 794 bp. PxLac2 encoded a protein with 597 amino acids and had a molecular weight of 66.09 kDa. Moreover, we determined the expression levels of PxLac2 in different stages by quantitative PCR (qPCR).The results indicated that PxLac2 was expressed differently in different stages. We observed the highest expression level in pupae and the lowest expression level in fourth instar larvae. We also investigated the enzymatic properties of laccase,which had optimal activity at pH 3.0 and at 35°C. Under these optimal conditions, laccase had a Michaelis constant (Km)of 0.97 mmol L-1, maximal reaction speed (Vm) of 56.82 U mL-1, and activation energy (Ea) of 17.36 kJ mol-1 to oxidize 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid ammonium salt). Type II copper enhanced laccase activity below 0.8 mmol L-1 and reduced enzyme activity above 0.8 mmol L-1 with an IC50 concentration of 1.26 mmol L-1. This study provides insights into the biological function of laccase.

Keywords: laccase, melanization, in vivo expression, enzymatic properties, Plutella xylostella

1. Introduction

Laccase (EC 1.10.3.2) is a member of the multicopper protein family and is known to oxidize various aromatic and nonaromatic compounds via a radical-catalyzed reaction(Strong and Claus 2011). Laccase was first discovered in lacquer trees (Rhus vernicifera) (Yoshida 1883) and is one of the widely studied glycoproteins that also has been used in other applications (Hüttermann et al. 2001). The Chinese have used the sap of the lacquer tree (polymerization function of the laccase) to create artwork for more than 6 000 years (Hüttermann et al. 2001). Laccase has been discovered in plants, animals, and microbes (Thomas et al.1989; Claus and Filip 1997). In insects, two types of laccase,Lac1 and Lac2, with different expression levels in different tissues have been reported (Dittmer et al. 2004). Lac1 is expressed mostly in the salivary gland, midgut, fat body,and malpighian tubule and plays a role in the oxidation of toxic compounds in the diet. Lac2 is expressed primarily in the cuticle, and plays a role in cuticular sclerotization and pigment synthesis (He et al. 2007; Niu et al. 2008). Mostlaccases are composed of three cupredoxin-like domains,including one type 1 (T1) copper (Cu1), one type 2 (T2)copper (Cu2), and two type 2 (T3) coppers (Cu3). Cu2 and Cu3 form a trinuclear center (Palmer et al. 2001; Garavaglia et al. 2004). A comparison of more than 100 laccase sequences has revealed the presence of four conserved motifs (L1-L4) in laccase (Kumar et al. 2003).

Lac2 is a highly conserved multicopper oxidase and is expressed in all developmental stages in insects (Arakane et al. 2005), and it has an important function in cuticular sclerotization and pigment synthesis (Arakane et al.2005; He et al. 2007; Gorman et al. 2008; Niu et al. 2008;Dittmer et al. 2009; Yatsu and Asano 2009; Elias-Neto et al. 2010; Futahashi et al. 2010). It is synthesized by the epithelial cells and is secreted to the locations where new epidermis formation takes place to fulfill its function of cuticle melanization (Dittmer et al. 2009; Yatsu and Asano 2009; Futahashi et al. 2010). Knockout of the Lac2 gene in Tribolium castaneum, Apis mellifera, Riptortus pedestris,and Drosophila melanogaster reduced melanization in the respective exoskeleton, leading eventually to death(Arakane et al. 2005; Niu et al. 2008; Elias-Neto et al. 2010;Futahashi et al. 2011; Riedel et al. 2011).

Laccase is difficult to purify from the insect exoskeleton and therefore the accurate catalytic constant (Kcat) of laccase is not known because of low purity and low amounts of the available enzyme. Most studies estimated the Michaelis constant (Km) of laccase to be between 0.2 and 8.7 mmol L-1(Yamazaki 1972; Andersen 1978; Barrett and Andersen 1981; Thomas et al. 1989; Dittmer et al. 2009). Insect laccases exist as inactive precursors, which are activated after molting. For example, inactive cuticle laccase extracts can be activated by trypsin (Yatsu and Asano 2009). Active laccase can oxidize a wide range of substrates, including polyphenols, methoxy-substituted phenols, aminophenols,and aromatic diamines (Baldrian 2006). Substrates bind to copper at the T1 site and substrate reduction is achieved by transferring electrons to the copper ion (Zhukhlistova et al. 2008).

Plutella xylostella L. (Lepidoptera: Plutellidae) is one of the most destructive pests of cruciferous vegetables.Because of its fast reproduction, overlapping generations,and pesticide abuse, P. xylostella has developed resistance to many pesticides. It is estimated that 400–500 million USD is used to control P. xylostella every year (Furlong et al.2013). Lac2 plays an important role in cuticle sclerotization in many insect species; however, there has been no such report about its function in P. xylostella until now. Thisstudy examines Lac2 properties in P. xylostella. We hope that our findings, such as blocking PxLac2 gene expression and disturbing the normal tanning of the diamondback moth cuticle, could provide a novel target to control P. xylostella in the future. On the basis of this significance, this study will determine the molecular and enzymatic characters of P. xylostella Lac2 (PxLac2) gene. We will obtain the cDNA sequence of PxLac2 followed by determining the expression levels of PxLac2 in different developmental stages and the enzymatic properties of Lac2. And we will investigate the effect of Cu2+on laccase activity. This study provides information about the physiological function of Lac2 in the insect exoskeleton.

2. Materials and methods

2.1. lnsects and reagents

We collected P. xylostella in the south campus field of Shandong Agricultural University, Shandong Province,China, in 2006. We reared the P. xylostella in a climate chamber at (25±1)°C, (60–70)% relative humidity and during a 14 h L:10 h D photoperiod by modifying a previously published protocol (Fang et al. 1988; Liu et al. 1993; Xu et al. 1997).

The RNAsimple Total RNA Kit, FastQuant RT Kit, and the quantitative real-time PCR (qPCR) reagent (SYBR Green) were from TIANGEN Biotech (Beijing, China). The SMARTer®RACE 5´/3´ Kit was from Clontech (Mountain View, CA, USA), and 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid ammonium salt (ABTS) was from Aladdin(Shanghai, China). Phenylmethanesulfonyl fluoride (PMSF)was from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were analytically pure.

2.2. Total RNA extraction and first-strand cDNA synthesis

We extracted total RNA from the third instar P. xylostella larvae using the RNAsimple Total RNA Kit following the manufacturer’s protocol. We used about 1.0 µg of extracted total RNA for first-strand cDNA synthesis using the FastQuant RT Kit following the manufacturer’s protocol.

2.3. Cloning of PxLac2 cDNA

We used the first-strand cDNA as the template in the rapid amplification of cDNA ends (RACE) protocol to identify the Lac2 open reading frame (ORF). We designed 5´and 3´ RACE primers using the predicted Lac2 protein(XM_011557273.1) at the National Center for Biotechnology Information (NCBI). We performed the RACE reactions using the SMARTer®RACE 5´/3´ Kit following the manufacturer’s protocol. We sequenced the amplified products and assembled the sequences using DNAMAN.We used ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder/) to predict the PxLac2 ORF. Primers used in this experiment are listed in Table 1.

2.4. PxLac2 sequence analysis

We used the NCBI’s Basic Local Alignment Search Tool(http://blast.ncbi.nlm.nih.gov/Blast.cgi) to search and compare sequences homologous with PxLac2. We used ProtParam (http://web.expasy.org/protparam/) to predict the isoelectric point (pI) and molecular weight (MW) of Lac2.We used SignalP (http://www.cbs.dtu.dk/services/SignalP)to identify the putative signal peptide. We compared Lac2 amino acid sequences from 21 different insects with the PxLac2 sequence using DNAMAN and constructed phylogenetic trees with the Neighbor-joining method using MEGA 7.0.

2.5. PxLac2 expression levels in different developmental stages

We extracted total RNA from the first to fourth instar larvae,prepupae, pupae, and adults. Then, we used 1.0 µg total RNA for cDNA synthesis, as described earlier. We used the resulting cDNA as a template in qPCR using the SYBR Green Kit (TIANGEN Biotech. Co., Ltd., Beijing). We performed qPCR in a 20-µL reaction volume containing 2-µL of the cDNA template, 10 µL of SuperReal PreMix Plus, and 0.6 µL each of the forward and reverse primers. We used two internal reference genes, RPS-13 (AY174891) and β-actin (AB282645), for qPCR to determine the Ctvalues.We used the mean value of these genes as the references and to compare PxLac2 relative expression. We carried out reactions on a Bio-Rad CFX-96 Real-time PCR System under the following conditions: 15 min at 95°C to activate polymerase followed by 40 cycles of denaturation at 95°C for 10 s, annealing at 55°C for 20 s, and elongation at 72°C for 20 s. We used the 2–ΔΔCTmethod to analyze the relative expression of PxLac2 in different developmental stages.

2.6. Laccase enzymatic properties

Laccase extractionWe performed laccase extraction following the protocol described by Yamazaki (1972) with slight modifications. First, we placed P. xylostella pupae on a plate and dissected the pupae vertically using a scalpel.We removed internal organs, cephalothorax, and excessive fat bodies and stored the remaining pupal exoskeleton at-70°C until further use. About 1.0 g of treated exoskeleton was ground and transferred to a 10-mL precooled centrifuge tube. We then washed the samples twice with 8 mL of 200 mmol L-1Tris-HCl.

We centrifuged Tris-HCl (pH 7.5) containing 6 mol L-1urea at 12 000×g at 4°C for 20 min. After washing, we discarded the supernatant and resuspended the pellets in the same Tris-HCl solution. Then, we added 28 mg trypsin to the samples, incubated the trypsin at 37°C for 2 h, and centrifuged it at 12 000×g at 4°C for 20 min. We transferred the supernatant to a new 10-mL tube and added ammonium sulfate until 50% saturation was reached. We placed the mixture at 4°C overnight and collected the protein precipitates by centrifuging at 12 000×g for 20 min at 4°C. Then, we used 500 µL of 10 mmol L-1Tris-HCl (pH 7.5, with 0.1 mmol L-1PMSF) to elute protein precipitates,which we dialyzed with 10 mmol L-1Tris-HCl until no sulfate was detected.

Laccase activity measurementWe determined laccase activity by measuring the oxidation of ABTS (Hu et al. 2001).We performed the assay in 96-well plates and each reaction was placed within a volume of 200 µL, containing a 140-µL reaction buffer, 20 µL of the laccase extract, and 40 µL of ABTS. To determine the optimal reaction conditions, we used different substrate concentrations (0.025, 0.05, 0.1,0.5 and 1 mmol L-1) and buffers at different pH conditions(2.2, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0) and incubated the reactions at various temperatures (20, 25, 30, 35, 40 and 45°C).We preheated substrates at the tested temperatures for 5 min before the actual assay. We measured the increase in absorbance spectrophotometrically (Epoch 2, BioTek,Winooski, VT, USA) for 2 min at 420 nm. We calculated enzyme activity based on Lambert-Beer’s law that the linear increase in optical density (ΔOD) changed over time.Each reaction had three replicates. We defined one activity unit as the amount of enzyme required to oxidize 1 µmol of ABTS in 1 min, which increased the ABTS free radical concentration. On the basis of the definition of an activity unit and the ABTS free radical concentration-absorption value coefficient (k=0.1844), the activity in units (U mL-1) was equal to 1 000×0.1844×ΔOD×dilution times (Zhang 2007).

Table 1 Primers used in this study

Laccase activation energy measurementRelative velocity (V0) of the enzyme reaction at pH 3.0 was firstdetermined at various substrate concentrations. We applied the Lineweaver-Burk plot to obtain the maximal reaction speed (Vm), which was determined at different temperatures.We drew the linearized Arrhenius curve by plotting the lnVmvs. 1/T. The slope of the Arrhenius plot indicated the activation energy of laccase for ABTS.

Effects of Cu2+ on laccase activityIn 200 µL reaction volumes, we added different concentrations of CuSO4(0.025, 0.05, 0.075, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1.0, 1.2 mmol L-1) to study the effect of various Cu2+concentrations on laccase activity as described above.

2.7. Data analysis

We statistically analyzed data using analysis of variance(ANOVA) and evaluated differences in the means by Tukey’s multiple comparison test (P＜0.05) by using SPSS 16.0(SPSS Inc., Chicago, IL, USA).

3. Results

3.1. Characterization of PxLac2 cDNA

The full-length PxLac2 cDNA was 1 944 bp long and contained a 1 794 bp ORF. We predicted that PxLac2 had a molecular weight of 66.09 kDa and a pI of 5.23 using ProtParam. PxLac2 had a half-life of 30 h and an instability coefficient of 32.52, indicating that Lac2 is a stable protein.The chemical formula of Lac2 was C2940H4589N789O887S28. We predicted that there was a cleavage site at the 21st amino acid and the first 20 amino acids formed a signal peptide by SignalP Software.

The predicted PxLac2 amino acid sequence showed 98% identity with predicted P. xylostella Lac2 in GenBank(XM_011557273.1). At the amino acid level, three copper binding sites were predicted at 67–177, 183–325, and 452–555 residues and PxLac2 contained a C-X-R-X-C sequence motif, which was highly conserved in insect laccase (Fig. 1).

3.2. PxLac2 homology comparison

By comparing the amino acid sequence of PxLac2 and homologous laccase from 21 other insect species, we identified a sequence motif that was specific to insect laccase (HWHG(X)9DG(X)5QCPI; Fig. 2). PxLac2 shared 61, 54, 56, and 63% homology with Amyelois transitella Lac1 (XP_013194946), Papilio polytes Lac2 (AB531135),Papilio xuthus Lac2 (AB499123), and Papilio machaon Lac2 (AB531133), respectively, indicating that PxLac2 was related closely to laccase from these insect species (Fig. 3).

3.3. PxLac2 expression in different developmentalstages

qPCR results showed that PxLac2 was expressed in all development stages, although at different expression levels.We defined the PxLac2 expression level in adults as 1.0.By comparing the Ctvalues of the average reference genes RPS-13 and β-actin, PxLac2 was found to have relatively high expression in the third instar larvae and pupae,with 3.41- and 4.84-fold higher than that of the adults,respectively, and PxLac2 was found to have low expression in the fourth instar larvae and prepupae, with 0.7- and 1.27-fold of the adults, respectively (Fig. 4).

3.4. Laccase enzymatic properties

Effects of pH and temperature on laccase activityWe determined the effect of pH on laccase activity at room temperature. The results showed that laccase had higher enzyme activity in acidic conditions and that the optimal pH was 3.0. At higher pH, laccase showed decreased enzyme activity (Fig. 5-A). We tested the effect of temperature on laccase activity under the optimal pH of 3.0. The results showed that temperature did not significantly affect the laccase activity and that the optimal temperature was 35°C(Fig. 5-B).

Determining the kinetic parameters of laccaseWe also evaluated the kinetic behavior of laccase on ABTS oxidation.Under the optimal pH and temperature conditions, ABTS oxidation by the enzyme followed Michaelis-Menten kinetics(Fig. 6, inset). The enzyme’s kinetic parameters, Kmand Vm,were 0.97 mmol L-1and 56.82 U mL-1, respectively (Fig. 6).

Fig. 1 The cDNA and encoded amino acid sequences of Lac2 from Plutella xylostella. Insect specific amino acid fragments are indicated by boxes. The copper fingers are indicated by lines.

Laccase activation energyTo measure the activation energy of laccase required to oxidize ABTS, we firstdetermined the V0at various concentrations. We determined the Vmat different temperatures and drew the linearized Arrhenius curve by plotting the lnVmvs. 1/T (Fig. 7). The slope of the Arrhenius plot indicated that the activation energy (Ea) of laccase for ABTS substrates was 17.36 kJ mol-1.

Fig. 3 Phylogenic tree based on the amino acid sequences of Lac2 from Plutella xylostella and other insects.

Effects of Cu2+ on laccase activityWe used various copper sulfate (CuSO4) concentrations to study the effect of Cu2+on laccase activity. The results showed that when the Cu2+concentration was lower than 0.8 mmol L-1laccase activity was enhanced, and that at 0.5 mmol L-1, Cu2+increased laccase activity by 40%. When Cu2+concentration was higher than 0.8 mmol L-1, Cu2+inhibited laccase activity(Fig. 8-A). The calculated IC50value of Cu2+was 1.26 mmol L-1. When laccase reacted with ABTS for 20 s, we added 0.5 and 1.2 mmol L-1Cu2+to the reaction system, respectively,and also monitored the course of activated or inhibited laccase activity (Fig. 8-B).

4. Discussion

Previous studies on laccase have focused primarily on microbial laccase and their potential for industrial applications (Cao et al. 2004). With the advancement of molecular biological techniques, information on insect laccase is being clarified, thus increasing its potential use in practical applications. In this study, we obtained the full-length cDNA sequence of PxLac2 using PCR and RACE. PxLac2 showed high similarity with other insect Lac2 at the amino acid level. Phylogenetic analysis based on Lac2 amino acid sequences showed that PxLac2 was closely related to P. polytes Lac2, P. xuthus Lac2, and P. machaon Lac2, sharing more than 50% homology with all three proteins. PxLac2 also has very high sequence homology with AtLac1 (XP_013194946), which may be due to the fact that AtLac1 was a predicted fragment, not a sequenced full-length nucleotide sequence. Additionally,although Lac1 and Lac2 have different functions, the two laccases were not clustered obviously to branches; thus,we believe that their different functions may be manifested by the helices and folds in the proteins’ tertiary structure,rather than in the proteins’ secondary structure. PxLac2 contained the laccase motif, HWHG(X)9DG(X)5QCPI, and the insect laccase motif, C-X-R-X-C. PxLac2 had three classic Cu-oxidase domains with a Cu2+in the catalytic center. The Cu2+plays an important role during the catalysis process (Claus et al. 2002). We predicted that the first 20 amino acids of PxLac2 to be the signal peptide by SignalP Software. Ribosomes use different signal peptides to attach to specific locations on the endoplasmic reticulum (ER).Signal peptides also are used to lead protein synthesis.After synthesis, the signal peptides are cleaved from the proteins and synthesized proteins are transported to ERs,where they are eventually secreted extracellularly to fulfill their function (Ye 1999). Therefore, this signal peptide may be involved in transporting PxLac2 to the exoskeleton where PxLac2 could be involved in melanization.

Fig. 4 Relative expression of PxLac2 in different developmentalstages of Plutella xylostella. 1L, 2L, 3L and 4L represent the 1st,2nd, 3th and 4th instar larvae. The relative expression value was calculated by the average of RPS-13 and β-actin, and the transcriptional expression of PxLac2 gene in the adult was set as 1.0. All the data were taken from a representative assay,representing the mean of three identical replicates±SD. The lowercase letters represent a significant difference (P＜0.05)between the different developmental stages.

Fig. 5 Effect of pH (A) and temperature (B) on Plutella xylostella laccase activity. All the data were taken from a representative assay, representing the mean of three identical replicates±SD. The lowercase letters represent a significant difference (P＜0.05)between the different pH (A) or temperature (B).

In this study, PxLac2 was expressed in all developmentalstages, although expression levels were different. PxLac2 had the highest expression level in the pupa stage. Insect pupae transform from larvae to adult insects, and in general,the melanization process is at the highest level during thisstage. Therefore, PxLac2 may play an important role in melanization and exoskeleton formation. Elias-Neto et al.(2010) showed that Lac2 from A. mellifera (AmLac2) had important functions in exoskeleton formation. In that study,AmLac2 was expressed earlier in the thoracic regions than in the abdominal integument, which matched the temporal order of melanization in the pupal exoskeleton. RNAi knockdown of AmLac2 resulted in the malformation of the exoskeleton and inhibited eclosion. The study also showed that AmLac2 was controlled by ecdysteroids and played an important role in adult exoskeleton differentiation. Blocking the release of ecdysteroids from thoracic to abdominal integument resulted in structural abnormalities in the exoskeleton (Elias-Neto et al. 2010). Lac2 from Aedes albopictus also had an important role in sclerotization and pigmentation. The expression level of AaLac2 increased after a blood meal in female A. albopictus, suggesting that Lac2 also may play a role in ovary maturation (Wu et al. 2013). In the study of Lac2 from Ostrinia furnacalis (OfLac2), Chen et al. (2014)found that the expression level of OfLac2 increased at the end of each larval stage and reached the highest level in the prepupal stage. OfLac2 had the highest expression level in the midgut and silk gland. Injecting the molting hormone,20-hydroxyecdysone (20E), into O. furnacalis upregulated OfLac2 12 h after injection and reached the highest level 24 h after injection, indicating that OfLac2 is regulated by 20E (Chen et al. 2014). Similarly, knockout of the 20E receptors, ecdysone receptor (EcR) and ultraspiracle (USP)isoform, in H. armigera reduced the levels of Lac2 (Cao et al. 2014).

Fig. 6 Lineweaver-Burk plots for the oxidation of ABTS by Plutella xylostella laccase. V, velocity; S, substrate.

Fig. 7 Arrhenius plots for the oxidation of ABTS by Plutella xylostella laccase. V, velocity; T, temperature (K).

Fig. 8 Effect of Cu2+ on Plutella xylostella laccase activity. A, the activation and inhibition of Cu2+ on laccase activity. B, the course of laccase-ABTS reaction. Curve 1 shows the course of reaction in the absence of Cu2+; curves 2 and 3 show the course of reaction in the presence of 0.5 and 1.2 mmol L-1 Cu2+ after 20 s, respectively.

The two isoforms of Lac2 in insects, Lac2A and Lac2B,likely are formed by alternative splicing (Arakane et al.2005). These isoforms exist in Bombyx mori, Manduca sexta, T. castaneum, Anopheles gambiae, Aedes aegypti,and D. melanogaster, but not in A. mellifera or Acyrthosiphon pisum (Gorman et al. 2008). Lac2A has been detected in the exoskeleton of B. mori, M. sexta, and A. gambiae (He et al. 2007; Dittmer et al. 2009; Yatsu and Asano 2009) but not Lac2B, indicating that Lac2B is not involved in insect sclerotization and melanization. Both isoforms (Lac2A and Lac2B) were involved in exoskeleton melanization in T. castaneum. Knocking down isoform 2A reduced the melanization level; knocking down isoform 2B delayed the start time of melanization; knockout of both isoforms severely hindered the melanization process (Arakane et al.2005). Both Lac2A and Lac2B from T. castaneum and A. gambiae did not show substrate specificity, indicating that both isoforms can oxidize the same substrates (Gorman et al.2012). We did not find evidence of alternative splicing of the Lac2 gene in the present study, and this needs further study.

In insects, although it is known that laccases catalyze dopamine (DA), N-acetyldopamine (NADA), N-βalanyldopamine (NBAD), 3,4-dihydroxyphenylalanine(DOPA), and catechol to their corresponding quinones(Thomas et al. 1989; Sugumaran et al. 1992; Dittmer et al.2009; Dittmer and Kanost 2010), little is known about the kinetic parameters of the enzyme in insects because cuticular laccases are difficult to purify (Gorman et al. 2012).Additionally, whether or not the laccases in P. xylostella are precursors, and the optimal pH at acidity or alkalinity, they pose problems that are important for the laccases catalytic reaction. In addition, given that laccases are a multicopperoxidase, the effects of heterologous Cu2+on laccase activity need to be investigated. Therefore, laccase property studies could shed light on enzyme functional exploration.

In this paper, we extracted crude laccase from P. xylostella pupae. Although inactive precursors were activated with trypsin in this study, the activation mechanisms are not known. Laccase showed high enzyme activity at low pH(optimal pH 3.0) and high temperature (optimal temperature 35°C). The optimal pH was similar to that of laccase in Nephotettix cincticeps (Hattori et al. 2005). Laccase had a Kmvalue of 0.97 mmol L-1under optimal pH and temperature, which was consistent with previous studies that Kmvalues ranged between 0.2 and 8.7 mmol L-1(Gorman et al. 2012). Low Cu2+concentration (＜0.8 mmol L-1)enhanced laccase enzyme activity, indicating that laccases are copper-containing enzymes.

In this study, we investigated the molecular and enzymatic properties of laccase; however, information about its function and gene regulation is still limited. Future studies should focus on understanding the function and gene regulation of PxLac2. Because laccase plays an important role in insect cuticle melanization, laccase could be used as a new target for pesticides to better control pest insects in the future.

5. Conclusion

In this paper, the cDNA of PxLac2 was cloned from the 3rd instar larvae in Plutella xylostella, and was also sequenced and characterized. The PxLac2 was differently expressed in different stages, the highest expression level was observed in pupae while the lowest expression level was in the 4th instar larvae. The enzymatic properties of laccase including optimal pH and temperature, Michaelis constant (Km) and the maximal reaction speed (Vm) were also determined. We found that Cu2+enhanced laccase activity below 0.8 mmol L–1and inhibited enzyme activity above 0.8 mmol L–1in the enzyme-substrate system.

Acknowledgements

This work was supported financially by the National Natural Science Foundation of China (31672046), the National Key Research and Development Program of China(2016YFD0200500), and the Funds of Shandong “Double Tops” Program, China (SYL2017YSTD06).