Research Article Cloning, Expression, and Characterization of Prophenoloxidases from Asian Corn Borer, Ostrinia Furnacalis (gunée)

Insect phenoloxidase (PO) belongs to the type 3 copper protein family and possesses oxidoreductase activities. PO is typically synthesized as a zymogen called prophenoloxidase (PPO) and requires the proteolytic activation to function. We here cloned full-length cDNA for 3 previously unidentified PPOs, which we named OfPPO1a, OfPPO1b, and OfPPO3, from Asian corn borer, Ostrinia furnacalis (Gunée), in addition to the previously known OfPPO2. These conceptual PPOs and OfPPO2 all contain two common copper-binding regions, two potential proteolytic activation sites, a plausible thiol-ester site, and a conserved C-terminal region but lack a secretion signal peptide sequence at the N-terminus. O. furnacalis PPOs were highly similar to other insect PPOs (42% to 79% identity) and clustered well with other lepidopteran PPOs. RT-PCR assay showed the transcripts of the 4 OfPPOs were all detected at the highest level in hemocytes and at the increased amounts after exposure to infection by bacteria and fungi. Additionally, we established an Escherichia coli (E. coli) expression system to produce recombinant O. furnacalis PPO proteins for future use in investigating their functions. These insights could provide valuable information for better understanding the activation and functioning mechanisms of O. furnacalis PPOs.


Materials and Methods
2.1. Insect Rearing. O. furnacalis larvae were reared on an artificial diet at 28 ∘ C under a relative humidity of 70-90% and a photoperiod of 16 h light and 8 h darkness [38].

Molecular Cloning of O. furnacalis PPO cDNAs.
Three transcripts for potential PPOs (CL5552.contig1, CL997.contig1, and Unigene28348) were previously identified from O. furnacalis transcriptome [20]. Among these, CL997.contig1 was a complete cDNA sequence, but another two transcripts (1,299-bp CL5552.contig1 and 1,152-bp Unigene28348) were incomplete. On the basis of these two fragments, primers (see Table S1 in Supplementary Material available at http://dx.doi .org/10.1155/2016/1781803) were designed for 5 and 3 rapid amplification of cDNA ends (RACE) to obtain the remaining sequences. The 5 and 3 RACE reactions were performed according to the manufacturer's instructions for its RACE kit (Takara, Biotechnology (Dalian) Co., Ltd., China) with cDNA from the whole body of fifth-instar O. furnacalis larvae collected 24 h after injection with 3 L of Micrococcus luteus (3 g/ L). Some modifications were made to obtain the 3end of CL5552.contig1. Briefly, after the first-round 3 RACE reaction using primer F1 (Table S1), a 1,566-bp fragment with 225 bp at 5 -end covered by CL5552.contig1 was obtained. It was then assembled into a 2,865-bp cDNA sequence ("PPO1assembled" in Figure 1(a)) combined with CL5552.contig1 (Figure 1(a)). However, two variant PCR products, which are both inconsistent with expected "PPO1-assembled," were amplified with selected primer pairs F3/R1 or F3/R2 (Figure 1(a), Table S1). They were designated as "PPO1a-1st" and "PPO1b-1st," respectively. The second-round 5 and 3 RACE reactions were performed for "PPO1a-1st" and "PPO1b-1st" following the manufacturer's instructions (Figure 1(a)). The resulting products were cloned and sequenced. After assembling, primers (Table S1) were designed to amplify the fulllength cDNA encompassing the entire reading frame using the same larval cDNA for RACE. The final PCR products ("PPO1a" and "PPO1b") were cloned into pMD19-T vector (Takara, Biotechnology (Dalian) Co., Ltd, China), and the nucleotide sequences were confirmed by DNA sequencing.

Sequence Analysis of O. furnacalis PPOs.
We carried out a series of sequence analyses for OfPPO1a, OfPPO1b, OfPPO2, and OfPPO3. The deduced amino acid sequences were obtained by using the Translate tool provided by the Swiss Institute Bioinformatics. Analysis of deduced amino acid sequences, including prediction of signal peptide, molecular weight, and isoelectric point, was executed in the EXPASY (Expert Protein Analysis System) proteomics server (http://www.expasy.org/). Characteristic domains or motifs were identified using DNAssist PROSITE profile database (http://prosite.expasy.org/scanprosite/).
To investigate the phylogenetic relationship between O. furnacalis PPOs and other arthropod PPOs, "prophenoloxidase" was used as a keyword to search the nonredundant database from the National Center for Biotechnology Information (NCBI; https://www.ncbi.nlm.nih.gov/). The retrieved PPO sequences were aligned with O. furnacalis PPO sequences using the Clustal W program. Phylogenetic trees were constructed by the neighbor-joining method using MEGA Version 5 software [39]. For neighbor-joining method, gaps were treated as characters, and statistical analysis was performed using the bootstrap method with 1000 replicates.   Figure 1: Cloning of OfPPO1a and OfPPO1b. (a) Schematic demonstration of the process for cloning full-length OfPPO1a (NCBI accession number: KX452359) and OfPPO1b (NCBI accession number: KX437622). The fragments with identical nucleotide sequences are illustrated in lines with the same color. The specific primers are labeled with the name followed by its position in the located fragment and indicated by the arrows above or below the lines. The predicted start codon and stop codon are indicated at the corresponding positions. (b) Comparisons and sequence analysis of deduced OfPPO1a and OfPPO1b. The deduced amino acid residues are shown in the same color scheme as in part (a). Identical residues in PPO1a and PPO1b are substituted by hyphens in PPO1b. Regions common in PPO1a and PPO1b are indicated by an irregular rectangle. The predicted proteolytic cleavage bonds were shown in red and marked with black arrow. The potential copper binding site A (CuA), copper binding site B (CuB), the putative thiol ester sites, and a conserved motif at the C-terminus were shaded in yellow, grey, green, and purple, respectively. The six histidine residues absolutely conserved within the CuA and CuB were in the square boxes. Two predicted disulfide linkages are represented by dashed lines.

Reverse Transcriptase-(RT-) PCR Analysis of the
Specific primers were designed and listed in Table S1. O. furnacalis ribosomal protein L8 (rpL8) was used as an internal standard to adjust the template amounts in preliminary PCR experiments. The thermal cycling conditions were 94 ∘ C for 2 min and then 28 cycles of 94 ∘ C for 30 s, 58 ∘ C for 30 s, and 72 ∘ C for 30 s followed by incubation at 72 ∘ C for 10 min. The PCR products were separated by electrophoresis on a 1.5% agarose gel.
To determine the expression patterns of O. furnacalis PPOs in different tissues, total RNA samples were isolated separately from combined heads, midguts, fat bodies, and hemocytes from 20-day-zero fifth-instar larvae. The synthesis of first-strand cDNA and RT-PCR analysis was performed as described above.
To check the expression of O. furnacalis PPOs after different microbial injections, day one fifth-instar larvae from the same batch were injected into the hemocele with 3 L of sterile water containing formalin-killed Escherichia coli DH5 (2 × 10 5 cells/ L), dried M. luteus (3 g/ L), Beauveria bassiana suspension (2 × 10 5 conidia/ L, B. bassiana conidia suspension was prepared as described previously [38]), or sterile water as a control. After 20 h (10 h for B. bassiana treatment), five larvae were collected from the challenged group and 5 from the control group, and total RNA samples were prepared from each larva. The following RT-PCR analysis was conducted as described above.

Production of Recombinant O. furnacalis PPO Proteins.
To express recombinant O. furnacalis PPOs in E. coli, the expression plasmids were constructed via FastCloning as described by Li et al. [40]. Briefly, the vector pET28a (Novagen,-Germany) and the coding region of mature OfPPO1a, OfPPO1b, OfPPO2, and OfPPO3 were amplified by PCR, respectively, using specific primers listed in Table S1. The forward primer for vector amplification (5 -CACCACCAC-CACCACCACTGAGAT-3 ) matched to the 3 side of the polylinker region of expression vector pET28a. The reverse primer for vector amplification (5 -GTGATGATGATGATG-ATGGCTGCT-3 ) was reversely complementary to the 5 side of the polylinker. The primers for OfPPO amplification had specific sequences and additional 15 nucleotides overlapping with the vector ends. The PCR products containing amplified vector and each OfPPO was mixed and digested with DpnI (Takara, Biotechnology (Dalian) Co., Ltd., China) for 1 h at 37 ∘ C. The digested mixture was then directly transformed into competent E. coli DH5 cells. After sequence verification, the resulting plasmids encoding correct OfPPOs were further transformed into E. coli strain BL21 (DE3) cells and plated onto Luria Bertani (LB) agar with kanamycin (0.05 mg/mL) for selection. For the coexpression of two OfPPOs, the constructed plasmids encoding OfPPO1a or OfPPO1b were individually transformed to E. coli BL21 (DE3) cells which already contained plasmids encoding OfPPO2 or OfPPO3, named as OfPPO1a and OfPPO2 (OfPPO1a/2), OfPPO1a and OfPPO3 (OfPPO1a/3), OfPPO1b and OfPPO2 (OfPPO1b/2), and OfPPO1b and OfPPO3 (OfPPO1b/3). Then single colony was picked and inoculated into 7 mL of LB medium with kanamycin (0.05 mg/mL) and incubated overnight at 37 ∘ C with constant shaking. The following day, 2 mL of the overnight cultures was inoculated into 100 mL of LB with kanamycin (0.05 mg/mL). When OD 600 of the culture reached 0.7, isopropyl thio--D-galactoside (IPTG) was added to a final concentration of 0.1 mM, and the recombinant PPO proteins were expressed for 12 h at 18 ∘ C. The bacteria were harvested by centrifugation at 10,000 g for 15 min and resuspended in lysis buffer (50 mM sodium phosphate, 300 mM NaCl, 10 mM imidazole, pH 8.0) to a uniform OD 600 to ensure equal protein loading. Cells were lysed by sonication and then centrifuged at 10,000 g for 30 min. The cleared supernatant was recorded as soluble fraction (which contained the soluble recombinant PPOs) while the cell debris was recorded as insoluble fraction (which contained the insoluble recombinant PPOs).

Activity Assay of Recombinant O. furnacalis PPO Proteins.
To check the PO activity of recombinant proteins, a convenient way was utilized as described by Li et al. [30]. Briefly, Cu 2+ was also added into the culture to a final concentration of 0.5 mM upon the addition of IPTG. Following the recombinant expression as described above, 1 mL of E. coli culture expressing each OfPPO was centrifuged at 10,000 g for 3 min. The resulting E. coli cells were washed using 10 mM Tris buffer (pH7.4) three times and then suspended in 30% ethanol for nonproteolytic activation of recombinant PPOs. After incubation at room temperature for 30 min, the reaction mixture was centrifuged at 10,000 g for 3 min. The cell pellet containing PPOs was then incubated with 250 L of dopamine (10 mM) for 5 min. Then PO activity of each sample was measured by monitoring A 490 in a microplate reader (Bio-Tek instrument, Inc.). One unit of PO activity was defined as the amount of enzyme producing an increase in absorbance (ΔA 490 ) of 0.001 per min.

Cloning and Analysis of O. furnacalis PPO cDNAs.
With "next generation" high-throughput sequencing, we obtained an O. furnacalis transcriptome dataset containing 62,382 unigenes [20]. Among these, three (1,299-bp CL5552.Contig1, 2,366-bp CL997.Contig1, and 1,153-bp Unigene28348) were predicted to encode potential PPOs [20]. The 2,366-bp CL997.Contig1 contains a complete 2,079-bp open reading frame and encodes a polypeptide sequence with 100% identity to O. furnacalis PPO (GenBank: ABC59699) previously reported by Feng et al. [37]. Here we designated it as OfPPO2. We performed 5 -and 3 -RACE to obtain the missing ends of the transcript CL5552.Contig1 and Unigene28348. After validation, CL5552.Contig1 was actually an assembly mosaic consisting of two variant sequences. The obtained full-length cDNA was nominated as OfPPO1a, OfPPO1b, and OfPPO3, respectively. They were submitted to NCBI and assigned the accession number as KX452359, KX437622, and KX437621, respectively. OfPPO1a and OfPPO1b matched with the 3and 5 -end of CL5552.Contig1, respectively (Figure 1(a)). We conducted PCR using the primers common in both OfPPO1a and OfPPO1b (Table S1) and subjected the PCR products for sequencing. Among 12 clones randomly selected, 6 ones were identical to OfPPO1a while the other 6 were identical to OfPPO1b. This indicated that OfPPO1a and OfPPO1b were expressed with the same abundance in O. furnacalis larvae.
The full-length cDNA of OfPPO1a, OfPPO1b, and OfPPO3 contains 2,462, 2,391, and 2,388 nucleotides, with a 2,052-, 2,058-, and 2,100-bp open reading frame, respectively (Figures 1 and 2). A poly(A) tail was found in the cDNA sequences of OfPPO1b and OfPPO3, but not in OfPPO1a. The conceptual proteins deduced from nucleotide sequences of OfPPO1a, OfPPO1b, and OfPPO3 cDNA consist of 683, 685, and 699 amino acid residues, respectively (Figures 1 and  2). The OfPPO2 cDNA encodes a polypeptide with 692 amino acid residues [37]. All four O. furnacalis PPOs, including the previous OfPPO2, lack a typical secretion signal peptide at their N-terminal regions. The calculated molecular mass and isoelectric point of the deduced proteins are 78.6 kDa and 6.59 for PPO1a, 78.6 kDa and 5.99 for PPO1b, 79.8 kDa and 5.72 for PPO2, and 81.0 kDa and 7.41 for PPO3, respectively.
Database search and sequence alignment indicated that 4 O. furnacalis PPOs exhibited high amino acid sequence similarity (42%-79% identity) with PPOs from other insect species: OfPPO1a, OfPPO1b, OfPPO2, and OfPPO3 are most similar in amino acid sequences to B. mori PPO1 (AAG09304), Mythimna separata PPO (BAM76811), Galleria mellonella PPO2 (AAQ75026), and G. mellonella PPO2 (AAQ75026), with the sequence identity of 74.5%, 74.1%, 79.3%, and 70.5%, respectively. A phylogenetic analysis was performed to investigate the evolutionary relationships between OfPPOs and other homologs. As shown in Figure 4, insect and crustacean PPOs form separate clades in the phylogenetic tree. The aligned insect PPOs (totally 89 PPOs from 34 insect species) are clustered into six distinct groups, including the subfamilies for two lepidopteran PPOs, two dipteran PPOs, one coleopteran PPO, and one hymenopteran PPO. The only inconsistency is Hemiptera PPO (Choristoneura fumiferana PPO2, ABW16862) is assigned to the group of lepidopteran PPOs. These six groups are divided into three major clades: Clade A consists of the conserved PPOs distributed among various insect orders, whereas Clades B and C comprise the paralogs specifically present in Lepidoptera and Diptera, respectively ( Figure 4). O. furnacalis PPO1a and PPO1b clustered with lepidopteran PPOs in Clade A and OfPPO2 and OfPPO3 grouped with lepidopteran PPOs in Clade B.

Expression Profiles of 4 PPO Transcripts in O. furnacalis.
We analyzed the mRNA levels of 4 O. furnacalis PPOs in various development stages, different tissues, and different pathogen challenges using semiquantitative RT-PCR methods. As shown in Figure 5, the four O. furnacalis PPOs exhibited distinct expression patterns. The OfPPO1a transcript was at the highest levels in eggs and the fifth-instar larval stage and then in the pupae. This transcript was barely detectable in the first to fourth-instar larval stage, especially in the first and second-instar larval stage. OfPPO1b and OfPPO2 were expressed in all tested developmental stages, including egg, larval, and pupal stages. However, OfPPO1b was expressed at the highest levels in eggs while OfPPO2 was expressed at similar levels in all stages. The OfPPO3 mRNA level increased from the second-instar larval stage, reached a maximum at   49  50  48  48  47  48  49  48  48   59  60  58  58  57  58  61  58  60   161  162  160  160  159  163  165  163  165   172  173  171  171  170  174  176  174  the fifth-instar stage, and decreased dramatically in the pupal stage ( Figure 5(a)). RT-PCR analyses showed that the 4 O. furnacalis PPO mRNAs were all detected in the hemocytes at the highest levels among the tested four tissues including heads, guts, hemocytes, and fat bodies. OfPPO1b mRNA was not detected in guts and fat bodies ( Figure 5(b)).

GGCGCCAAATACGTGCTCTTCGTCATGCTGTCCAACTATGAAGGGGATAGGGTGGCGCAAGCTAACGTACATCGCGAGTTGACGTGC GACGAGGGCGGCCGTGCCTGGCTTCTCTCCGACCAGCGCAAGATGTTCATCGAGATGGACAAGTTTGTTCACGACTTACGACCTGGT D E G G R A W L L S D Q R K M F I E M D K F V H D L R P G D P G D P N N L E F N Y C G C G W P Q H M L L P K G T A A G A K Y V L F V M L S N Y E G D R V A Q A N V H R E L T C AAAGAGGCGTCAAGCTTCTGCGGCTTGAGAGACCGCCTGTACCCGGACAAGCGCGCGATGGGATTCCCCTTCGACCGGCCGTCCACC K E A S S F C G L R D R L Y P D K R A M G F P F D R P S T ACCGCCGACCACATCGACGACTTCCTAACCCGCAACATGTTCGTGCAGGACGTCACCATCCGCTTCAGTGACGTCACCGAACAGAAC T A D H I D D F L T R N M F V Q D V T I R F S D V T E Q N
To  (Figure 5(c)).   were under control of strong bacteriophage T7 transcription and translation signals and their expression was induced by providing a source of T7 RNA polymerase in the host BL21 (DE3) cells. Using a highly simplified and efficient PCR-based cloning technique [40], the complete open reading frame of 4 O. furnacalis PPOs was successfully inserted into the pET28a vector individually. In the constructed plasmids, two 6 × His tags were added into the amino-and carboxyl-terminus of recombinant PPO proteins, respectively ( Figure S1). Additionally, we successfully transformed dual plasmids into BL21 (DE3) cells in order to express pairs of PPOs simultaneously, including OfPPO1a and OfPPO2, OfPPO1a and OfPPO3, OfPPO1b and OfPPO2, and OfPPO1b and OfPPO3 ( Figure S3A). The recombinant expression of 4 O. furnacalis PPOs was evaluated at different temperatures and induction conditions to determine the optimum parameters for protein expression (data not shown). Eventually, three of four O. furnacalis PPOs (OfPPO1b, OfPPO2, and OfPPO3, but not OfPPO1a) were successfully expressed under the IPTG induction ( Figure 6(a)). Although the insect-produced PPOs are soluble proteins [24,41,42], large proportions of recombinant OfPPO1b, OfPPO2, and OfPPO3 formed insoluble aggregates in the E. coli cells even using a low concentration of IPTG (0.1 mM) and a relatively low temperature (18 ∘ C). These aggregates fractionated with the cell debris following lysis (Figure 6(a)). Under the same induction and lysis conditions, the solubility of recombinant OfPPO2 was obviously higher than that of recombinant OfPPO1b and OfPPO3. The soluble fractions of recombinant OfPPO1b and OfPPO3 were nearly undetectable in polyacrylamide gel electrophoresis (Figure 6(a)).

Production of Recombinant O. furnacalis PPOs in the
Additionally, recombinant OfPPO1b, OfPPO2, and OfPPO3, but not OfPPO1a, could be recognized by antibody against His-tag ( Figure 6(b)). The cultured cells were treated with 30% ethanol for activating recombinant OfPPOs as described by Li et al. [30]. The resulting PO activity was checked using dopamine as substrate. As Figure 6(c) shown, four ethanol-treated OfPPOs with the exception of OfPPO1a exhibited detectable PO activities. There was no significant difference among the PO activity of recombinants OfPPO1b, OfPPO2, and OfPPO3.

Discussion
Phenoloxidase (PO) is the important enzyme in the melanin synthesis, a pathway in which some secondary reaction products including reactive oxygen species (ROS) and reactive OfPPO1b OfPPO2 OfPPO3 Anti-His The cell lysate was prepared as described in "Materials and Methods and incubated with 30% ethanol for activation [30]. PO activity of each protein sample was measured with dopamine as substrate. The bars represent mean ± SD ( = 3). Bars labeled with different letters (a or b) are significantly different (one-way ANOVA, followed by the Newman-Keuls test, < 0.05).
nitrogen species (RNS) are also generated [3,11]. PO plays a key role in multiple physiological processes including wound healing [44], hemolymph clotting [45], and innate immune response especially in defensive encapsulation and melanization of foreign organisms [11,46]. In this study, we cloned full-length nucleotide sequences for 3 previously unknown O. furnacalis PPOs (OfPPO1a, OfPPO1b, and OfPPO3) together with OfPPO2, which had been reported previously [37]. We investigated the expression profiles of these 4 PPOs and performed the recombinant expression using the prokaryotic expression system.
The three previously undocumented full-length cDNA sequences for O. furnacalis PPOs encode polypeptides with ∼700 amino acid residues and apparent molecular weight of ∼ 80 kDa. As described in other insects [41,47], the conceptual O. furnacalis PPO proteins also contain four conserved regions including two possible copper binding sites, one thiol ester-like motif and one common C-terminal end ( Figures  1-3). The copper binding sites with six absolutely conserved histidine residues are common in all arthropod POs and other closely related proteins such as arthropod hemocyanins [47]. They play an important role in encapsulation of foreign particles [48]. O. furnacalis PPOs are potentially responsible for oxygen transport and melanization process due to the presence of these two binding sites. The high similarity and the shared conserved domains of O. furnacalis PPOs and arthropod PPOs infer that the identified O. furnacalis PPOs in this study are members of the PO family.
In addition to the structurally necessary domains and motifs mentioned above, there are other two aspects worthy of note in O. furnacalis PPOs. Firstly, no typical secretion signal peptide was predicted at the N-terminal region of the deduced O. furnacalis PPO1a, PPO1b, and PPO3 (Figures 1  and 2). This is also consistent with the case in O. furnacalis PPO2 [37] and other insect PPOs excluding venom PPO in the parasitoid wasp, Pimpla hypochondriaca [11,19,49]. In D. melanogaster, PPO was demonstrated to be released by the rupture of crystal cells [21]. In other arthropods, there are possibly other ways to release PPO than by cell rupture [11,50]. So far, it is unclear whether and how O. furnacalis PPOs are released into the extracellular milieu. Secondly, the released PPO exists as zymogen in the hemolymph and must be activated by proteolytic cleavage [11,20]. The presumed cleavage and activation sites of 4 O. furnacalis PPOs were predicted between Arg 52 and Phe 53 in OfPPO1a, Arg 53 and Phe 54 in OfPPO1b, Arg 51 and Phe 52 in OfPPO2, and Arg 52 and Phe 53 in OfPPO3, as previously reported [13,16,46]. In H. diomphalia, HdPPO was cleaved at the conserved bond Arg 50 -Phe 51 , followed by the second cleavage at Arg 162 -Ala 163 [23,24]. In the housefly Musca domestica, the activation of PPO may occur after the cleavage at Arg 164 -Glu 165 [51]. The Arg-Ala bond in HdPPO was identified at the common position in OfPPO1a and OfPPO1b, but not in OfPPO2 and OfPPO3. The Arg-Glu bond in MdPPO was consistently present in O. furnacalis PPOs except for OfPPO3 with Arg-Thr instead (Figure 3(a)). We have determined two serine proteases, SP13 and SP105, which functioned as prophenoloxidase-activating proteases to cleave and activate OfPPO2 [52]. However, the precise activation site(s) of O. furnacalis PPOs is still unknown. It is also unclear whether or not there exist other possible cleavage sites other than the typical Arg-Phe bond.
Insect POs are artificially divided into tyrosinase-type POs and laccase-type POs in some reports: the former can hydroxylate monophenols to o-diphenols (EC 1.14.18.1) and then oxidize the o-diphenols to quinones (EC 1.10.3.1), but it cannot oxidize p-diphenols; the latter lack the monophenol monooxygenase activity but can oxidize both o-and pdiphenols (EC 1.10.3.2) [53,54]. A phylogenetic analysis of the 4 O. furnacalis PPOs with insect tyrosinase-type and laccase-type POs revealed two distinct clades with 100% bootstrap support. All 4 O. furnacalis PPOs fell inside the cluster of tyrosinase-type POs ( Figure S2). This suggests that the identified O. furnacalis PPOs are orthologous to tyrosinases rather than laccases. The precise classification of O. furnacalis PPOs cannot be determined until their activities toward monophenols and p-diphenols are analyzed. Furthermore, the phylogenetic relationship among O. furnacalis PPOs and other arthropod tyrosinase-type POs was proposed in this work. Lepidopteran, dipteran, coleopteran, and hymenopteran PPOs were separated in distinct clusters. Lepidopteran and dipteran PPOs each fell into two separate groups. O. furnacalis PPO1a and PPO1b were clustered in one lepidopteran PPO group while PPO2 and PPO3 were in the other lepidopteran PPO group (Figure 4). It suggests that lepidopteran PPO genes including O. furnacalis PPOs may have undergone duplication and divergence during evolution.
It is of particular interest that up to 4 O. furnacalis PPO sequences are identified in this study since only two PPO genes have been reported in most lepidopteran species [13,16]. These 4 O. furnacalis PPOs were differentially expressed with respect to developmental stages, tissues, or induced conditions ( Figure 5). Even PPO1a and PPO1b, which are 86.82% identical in deduced amino acid sequences, also exhibited distinct expression patterns. For example, PPO1a mRNAs were detected in all tested tissues including head, gut, hemocytes, and fat bodies while OfPPO1b transcripts were barely detectable in gut and fat bodies ( Figure 5(b)). Moreover, recombinant OfPPO1b, OfPPO2, and OfPPO3 could be produced successfully in prokaryotic expression system and be activated by ethanol. OfPPO1a failed to be expressed in E. coli ( Figure 6). Three PPO genes were also identified in the swallowtail butterfly, Papilio xuthus ( Figure 4) [55]. Therefore, we infer that all identified PPOs indeed exist and potentially perform different functions in O. furnacalis, although we currently do not know why O. furnacalis possesses more PPO genes than other lepidopterans. Explanations for the presence of 4 PPO sequences will be possible only when more data on PO function in O. furnacalis are obtained. Obtaining the recombinant PPO proteins in vitro in this study was just the first step to this aim. The 3 O. furnacalis PPOs expressed in E. coli were observed to have obvious enzyme activities after activation by ethanol ( Figure 6(c)). In order to potentially increase the solubility, we also tried to obtain PPO heterodimer by performing the coexpression, but failed possibly due to plasmid instability or incompatibility ( Figure S3). In future work, we will attempt to produce the recombinant PPOs with higher activity and then investigate their activation mechanisms and detailed functions.