Research Article Cloning, Expression, and Characterization...

Research ArticleCloning Expression and Characterization of Prophenoloxidasesfrom Asian Corn Borer Ostrinia furnacalis (Guneacutee)

Shasha Zhang Fang Hong He Song Lei Wang Qizhi Liu and Chunju An

Department of Entomology China Agricultural University Beijing 100193 China

Correspondence should be addressed to Chunju An anchunjucaueducn

Received 27 July 2016 Revised 27 September 2016 Accepted 26 October 2016

Academic Editor Daniela Cosentino-Gomes

Copyright copy 2016 Shasha Zhang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Insect phenoloxidase (PO) belongs to the type 3 copper protein family and possesses oxidoreductase activities PO is typicallysynthesized 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 borerOstrinia furnacalis (Gunee) in addition to the previously known OfPPO2 These conceptual PPOs and OfPPO2 all contain twocommon copper-binding regions two potential proteolytic activation sites a plausible thiol-ester site and a conserved C-terminalregion 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 OfPPOswere all detected at the highest level in hemocytes and at the increased amounts after exposure to infection by bacteria and fungiAdditionally we established an Escherichia coli (E coli) expression system to produce recombinant O furnacalis PPO proteins forfuture use in investigating their functionsThese insights could provide valuable information for better understanding the activationand functioning mechanisms of O furnacalis PPOs

1 Introduction

Reactive oxygen species (ROS) are chemically reactivemolecules that contain oxygen [1 2] Endogenous ROS areproduced as byproducts during aerobic respiration or byoxidation reactions catalyzed by metal and oxidoreductaseenzymes such as phenoloxidases (POs) [3 4] POs togetherwith tyrosinases catechol oxidase and hemocyanin belongto the type 3 copper protein family all of which have an activesite composed of two histidine-coordinated copper atoms [56] PO possesses both monophenol monooxygenase activity(EC 114181 tyrosine dihydroxyphenylalanine oxygen andoxidoreductase) and o-diphenoloxidase activity (EC 11031o-diphenol oxygen and oxidoreductase) [7 8] ThereforePOs can catalyze the hydroxylation of monophenols too-diphenols and subsequent oxidation of o-diphenols tocorresponding highly reactive o-quinones [9] PO-generatedquinones act as cross-linkers for wound healing or polymer-ize to form melanin capsules around the invading parasitesand parasitoids [10 11] or interact with oxygen to form ROSto kill microbial pathogens directly [4 12]

Although type 3 copper proteins are distributed amongalmost all organisms POs are mainly found in arthropodsincluding insects [6 13] In insects the number of PO genesranges from ten in themosquitoAedes aegypti [14] to just onein the honeybeeApismellifera [15] Inmost Lepidoptera thereare two PO genes [16] For example two PO sequences havebeen reported in the silkworm Bombyx mori [17] the beetleHolotrichia diomphalia [18] and the tobacco hornwormManduca sexta [19] However three transcripts for potentialPO were identified in the transcriptome of Asian corn borerOstrinia furnacalis (Gunee) [20]

PO is predominantly synthesized in hemocytes as azymogen called prophenoloxidase (PPO) and released intohemolymph (insect blood) to perform functions [16] How-ever all arthropod PPOs studied so far lack a signal peptidefor secretion into hemolymph [11 16] PPO is believedto be released after rupture of hemocytes [21 22] Theninactive PPO is converted into active PO form by proteolyticcleavage in hemolymph [23ndash25] The cleavage site in PPOhas been experimentally confirmed in several insect speciesincluding 51Arg-Phe52 in B mori PPO [26] 51Arg-Phe52 in

Hindawi Publishing CorporationJournal of Immunology ResearchVolume 2016 Article ID 1781803 13 pageshttpdxdoiorg10115520161781803

2 Journal of Immunology Research

M sexta PPO [27 28] and 50Arg-Phe51 followed by thesecond cleavage at 162Arg-Ala163 in H diomphalia PPO [2324] In vitro PPO can also be activated without proteolyticcleavage by certain chemicals such as ethanol or detergents(eg sodium dodecyl sulfate (SDS) cetylpyridinium chloride(CPC)) [29 30]

The activation of PPO is necessary for PO tofunction A model has generally been accepted for PPOactivation in insects upon pathogenic infections orphysical injuries a serine protease cascade is triggeredto activate the prophenoloxidase-activating protease andprophenoloxidase-activating protease then cleaves PPO at aconserved peptide bond to produce PO [11 20] This processinvolves multiple proteins including pattern recognitionproteins [31] a series of serine proteases [32] serine proteasehomologs [33] and serine protease inhibitors [34]

TheAsian corn borerO furnacalis (Gunee) (LepidopteraCrambidae) is an important insect pest in Asia causingserious damage to corn sorghum and millet [35] Howeverknowledge about PPO in O furnacalis is very incompleteFeng et al purified a PPO from O furnacalis hemolymph[36] and cloned its encoding cDNA [37] Our previouswork identified three transcripts for potential PPOs in thetranscriptome of this pest [20] Among these only onedesignated as OfPPO2 contained a complete open read-ing frame [20 37] In this study we cloned a full-lengthnucleotide sequence for another two O furnacalis PPOswhich we calledOfPPO1 andOfPPO3 (GenBank KX437621)OfPPO1 from the transcriptome is actually an assemblymosaic consisting of two variant PPO sequences OfPPO1a(GenBank KX452359) and OfPPO1b (GenBank KX437622)which are 8682 identical in amino acid sequences Weinvestigated the expression profiles of these 4 PPOs indifferent developmental stages in different tissues and uponthe infection of different microorganisms Additionally weperformed the recombinant expression for these 4 proteinsusing a prokaryotic expression system

2 Materials and Methods

21 Insect Rearing O furnacalis larvae were reared on anartificial diet at 28∘Cunder a relative humidity of 70ndash90 anda photoperiod of 16 h light and 8 h darkness [38]

22MolecularCloning ofO furnacalis PPO cDNAs Three tran-scripts for potential PPOs (CL5552contig1 CL997contig1and Unigene28348) were previously identified from O fur-nacalis transcriptome [20] Among these CL997contig1 wasa complete cDNA sequence but another two transcripts(1299-bp CL5552contig1 and 1152-bp Unigene28348) wereincomplete On the basis of these two fragments primers (seeTable S1 in Supplementary Material available at httpdxdoiorg10115520161781803) were designed for 51015840 and 31015840 rapidamplification of cDNA ends (RACE) to obtain the remainingsequences The 51015840 and 31015840 RACE reactions were performedaccording to the manufacturerrsquos instructions for its RACEkit (Takara Biotechnology (Dalian) Co Ltd China) withcDNA from the whole body of fifth-instarO furnacalis larvaecollected 24 h after injection with 3 120583L of Micrococcus luteus

(3 120583g120583L) Some modifications were made to obtain the 31015840-end of CL5552contig1 Briefly after the first-round 31015840 RACEreaction using primer F1 (Table S1) a 1566-bp fragment with225 bp at 51015840-end covered by CL5552contig1 was obtained Itwas then assembled into a 2865-bp cDNA sequence (ldquoPPO1-assembledrdquo in Figure 1(a)) combined with CL5552contig1(Figure 1(a)) However two variant PCR products whichare both inconsistent with expected ldquoPPO1-assembledrdquo wereamplified with selected primer pairs F3R1 or F3R2 (Fig-ure 1(a) Table S1) They were designated as ldquoPPO1a-1strdquo andldquoPPO1b-1strdquo respectively The second-round 51015840 and 31015840 RACEreactions were performed for ldquoPPO1a-1strdquo and ldquoPPO1b-1strdquofollowing the manufacturerrsquos instructions (Figure 1(a)) Theresulting products were cloned and sequenced After assem-bling primers (Table S1) were designed to amplify the full-length cDNA encompassing the entire reading frame usingthe same larval cDNA for RACE The final PCR products(ldquoPPO1ardquo and ldquoPPO1brdquo) were cloned into pMD19-T vector(Takara Biotechnology (Dalian) Co Ltd China) and thenucleotide sequences were confirmed by DNA sequencing

23 Sequence Analysis of O furnacalis PPOs We carriedout a series of sequence analyses for OfPPO1a OfPPO1bOfPPO2 and OfPPO3 The deduced amino acid sequenceswere obtained by using the Translate tool provided by theSwiss Institute Bioinformatics Analysis of deduced aminoacid sequences including prediction of signal peptidemolec-ular weight and isoelectric point was executed in theEXPASY (Expert Protein Analysis System) proteomics server(httpwwwexpasyorg) Characteristic domains or motifswere identified using DNAssist PROSITE profile database(httpprositeexpasyorgscanprosite)

To investigate the phylogenetic relationship betweenO furnacalis PPOs and other arthropod PPOs ldquoprophe-noloxidaserdquo was used as a keyword to search the nonre-dundant database from the National Center for Biotech-nology Information (NCBI httpswwwncbinlmnihgov)The retrieved PPO sequences were aligned with O furnacalisPPO sequences using the Clustal W program Phylogenetictrees were constructed by the neighbor-joining methodusing MEGA Version 5 software [39] For neighbor-joiningmethod gaps were treated as characters and statisticalanalysis was performed using the bootstrap method with1000 replicates

24 Reverse Transcriptase- (RT-) PCR Analysis of the Expres-sion Profiles of O furnacalis PPOs To investigate the changesof O furnacalis PPO transcript levels in several develop-mental stages total RNA samples were individually prepared(119899 = 5) from three different stages including egg larvae andpupa using TRNzol Reagent (TIANGEN Biotech (Beijing)Co Ltd China) 1 120583g of RNA samples equally from 5individual RNA samples in each stage was treated withDNase I (TIANGEN Biotech (Beijing) Co Ltd China)and converted into first-strand cDNA from an oligo (dT)primer following the instructions for QuantScriptRT Kit(TIANGEN Biotech (Beijing) Co Ltd China) The cDNAproducts independently from 3 biological replicates werediluted 10-fold for use as template in RT-PCR experiments

Journal of Immunology Research 3

CL5552Contig1 ATG 79

F1-889

RACE(F1)

1300

TAG

2131 ATG PPO1 - Assembled TAG

PPO1a -1st TAG

Assembled

Assembled

ATG

110ATG TAG

2107

2159

36ATG TAG

2085

PCR (F4R1)

ATG3

TAA2184

PCR (F2R2 followed by F3R1)

TAGATG

ATG TAG129 2184

ATG TAG3 2058

F2-9

F3-77

R1-2414

R2-2517

R3-1101RACE(R3)

R1-2462

F4-75

F5-1244R5-231RACE(R5) RACE(F5)

Assembled

F3-127 R6-2220PCR (F3R6)

PCR (F2R2followed byF3R2)

PPO1a - 2nd

PPO1a

PPO1b - 1st

PPO1b - 2nd

PPO1b

(1299bp)

(2865bp)

(2410 bp)

(2462bp)

(2383 bp)

(2366bp)

(2094bp)

(2499bp)

126 bp

217 bp

731 bp 412 bp

415 bp231 bp

411 bp

225 bp

884 bp

864bp

1223 bp

1566 bp

1135 bp

98bp

3998400

5998400

5998400 3

998400

(a)

MAD 68EARRNLMLFFDRPTEPCFMQKGDDKTVFQLPDHYYPDKYKALSTTLSDRFGTDDARIVQVSNIGL---A--KK--L------------------------E-------R----------------V-P-A---- 69

PPO1aPPO1b

PDLSLPKQLPYNEQFSLFIQKHREMAGSLIDTLVGMRNIEDLTSVCSYCQLHINPYMFNYCLAVALLHR 137----V-M--------------------N----F-----V-----L-T----R----------s------ 138DDTKGFNVPTVVQTFPDKFMDPKVFRRAREVSNVVTTGPRMPVVIEQNYTASDAEPEQRVAYFREDIGI 206----------L---------------K-----------S----E-PT-L---EL--------------V 207NLHHWHWHLVYPFEADFAIVNKDRRGELFYYMHQQIIARYNVERFCNGLGRVERYTNFRAPIAEAYFPK 275--------------T-------------L-----------TS----------V---------E------ 276LDSQVASRAWPPRFAGSILRDLDRPVDRIRIEVSELERWRDRFLQAIEENAVLVPGNRKVPLTEETGID 344-----------------V------------------------V---------I-S-------------- 345VLGNLMESSILSRNRGYYGDLHNMGHVFCSYAHDPDHRHLEQYGVMGDSATAMRDPFFYRWHAYVDDIF 413------------P--------------I---------------------T-------------F---V- 414NMHKVKLPKYGADRLDFPGIRVSSISVEGPAGRNTFGTQWEQSTVDLARGLDFTPRGSVLAQFTHLTHE 482D---T------N--------------------S----------M-----------------K----Q-D 483EFTYVIEVNNSSGRSTTGMFRIFIAPVNDDRGQPLSFADQRRLFIELDKFSQPLNTGNNTVRRSSVDSS 551----------T--AAAM------L-----ES----R-EE----------------A----I--N----- 552VTIPYERTFMDQTKRPGDPGSATAAEFDFCGCGWPHHMLIPKGTARGYPMVLFVMVSNWNDDRILQDTS 620-------------A--------A--------------L------PQ-F---------------VE---V 621GSCNDAASYCGLRDRKYPDRRAMGFPFDRPPQASTLSSFLRPNMAIRPCSVRFTDQVRIRPQQ 683-T---------I-------K------------E----D------S--Q-T-K---At---Q-RR 685

PPO1aPPO1bPPO1aPPO1bPPO1aPPO1bPPO1aPPO1bPPO1aPPO1bPPO1aPPO1bPPO1aPPO1bPPO1aPPO1bPPO1aPPO1b

S S S S

(b)

Figure 1 Cloning of OfPPO1a and OfPPO1b (a) Schematic demonstration of the process for cloning full-length OfPPO1a (NCBI accessionnumber KX452359) and OfPPO1b (NCBI accession number KX437622) The fragments with identical nucleotide sequences are illustratedin lines with the same color The specific primers are labeled with the name followed by its position in the located fragment and indicated bythe arrows above or below the linesThe predicted start codon and stop codon are indicated at the corresponding positions (b) Comparisonsand 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 anirregular rectangle The predicted proteolytic cleavage bonds were shown in red and marked with black arrow The potential copper bindingsite A (CuA) copper binding site B (CuB) the putative thiol ester sites and a conserved motif at the C-terminus were shaded in yellowgrey green and purple respectively The six histidine residues absolutely conserved within the CuA and CuB were in the square boxes Twopredicted disulfide linkages are represented by dashed lines

Specific primers were designed and listed in Table S1 Ofurnacalis ribosomal protein L8 (rpL8) was used as an internalstandard to adjust the template amounts in preliminary PCRexperiments The thermal cycling conditions were 94∘C for2min and then 28 cycles of 94∘C for 30 s 58∘C for 30 s and72∘C for 30 s followed by incubation at 72∘C for 10min The

PCR products were separated by electrophoresis on a 15agarose gel

To determine the expression patterns of O furnacalisPPOs in different tissues total RNA samples were isolatedseparately from combined heads midguts fat bodies andhemocytes from 20-day-zero fifth-instar larvaeThe synthesis


of first-strand cDNA and RT-PCR analysis was performed asdescribed above

To check the expression of O furnacalis PPOs afterdifferentmicrobial injections day one fifth-instar larvae fromthe same batch were injected into the hemocele with 3120583Lof sterile water containing formalin-killed Escherichia coliDH5120572 (2 times 105 cells120583L) driedM luteus (3 120583g120583L) Beauveriabassiana suspension (2 times 105 conidia120583L B bassiana conidiasuspension was prepared as described previously [38]) orsterile water as a control After 20 h (10 h for B bassianatreatment) five larvae were collected from the challengedgroup and 5 from the control group and total RNA sampleswere prepared from each larva The following RT-PCRanalysis was conducted as described above

25 Production of Recombinant O furnacalis PPO ProteinsTo express recombinant O furnacalis PPOs in E coli the ex-pression plasmids were constructed via FastCloning as de-scribed by Li et al [40] Briefly the vector pET28a (Novagen-Germany) and the coding region of mature OfPPO1aOfPPO1b OfPPO2 and OfPPO3 were amplified by PCRrespectively using specific primers listed in Table S1 Theforward primer for vector amplification (51015840-CACCACCAC-CACCACCACTGAGAT-31015840) matched to the 31015840 side of thepolylinker region of expression vector pET28a The reverseprimer for vector amplification (51015840-GTGATGATGATGATG-ATGGCTGCT-31015840) was reversely complementary to the 51015840side of the polylinker The primers for OfPPO amplificationhad specific sequences and additional 15 nucleotides over-lapping with the vector ends The PCR products containingamplified vector and each OfPPO was mixed and digestedwith DpnI (Takara Biotechnology (Dalian) Co Ltd China)for 1 h at 37∘C The digested mixture was then directly trans-formed into competent E coli DH5120572 cells After sequenceverification the resulting plasmids encoding correct OfPPOswere further transformed into E coli strain BL21 (DE3) cellsand plated onto Luria Bertani (LB) agar with kanamycin(005mgmL) for selection For the coexpression of twoOfPPOs the constructed plasmids encoding OfPPO1a orOfPPO1bwere individually transformed toE coliBL21 (DE3)cells which already contained plasmids encoding OfPPO2or OfPPO3 named as OfPPO1a and OfPPO2 (OfPPO1a2)OfPPO1a andOfPPO3 (OfPPO1a3) OfPPO1b andOfPPO2(OfPPO1b2) and OfPPO1b and OfPPO3 (OfPPO1b3)Then single colony was picked and inoculated into 7mL ofLB medium with kanamycin (005mgmL) and incubatedovernight at 37∘C with constant shaking The following day2mL of the overnight cultures was inoculated into 100mLof LB with kanamycin (005mgmL) When OD

600of the

culture reached 07 isopropyl thio-120573-D-galactoside (IPTG)was added to a final concentration of 01mM and therecombinant PPO proteins were expressed for 12 h at 18∘CThe bacteria were harvested by centrifugation at 10000 gfor 15min and resuspended in lysis buffer (50mM sodiumphosphate 300mM NaCl 10mM imidazole pH 80) to auniform OD

600to ensure equal protein loading Cells were

lysed by sonication and then centrifuged at 10000 g for30min The cleared supernatant was recorded as solublefraction (which contained the soluble recombinant PPOs)

while the cell debris was recorded as insoluble fraction (whichcontained the insoluble recombinant PPOs)

26 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)and Immunoblot Analysis For SDS-PAGE protein sam-ples were treated with 2 times SDS sample buffer containingDithiothreitol (DTT) at 95∘C for 5min and then separatedby 10 SDS-PAGE Proteins were detected by stainingwith Coomassie brilliant blue For immunoblot analysisproteins were transferred onto a nitrocellulose membraneand detected with mouse anti-His (1 2000) as primaryantibody Antibody binding was visualized using alkalinephosphatase-conjugated goat anti-mouse or anti-rabbit IgG(1 1500) and 5-bromo-4-chloro-3-indolyl phosphatenitroblue tetrazolium (BCIPNBT) staining buffer containing165 120583gmLBCIP and 330 120583gmLNBT in 100mMTris (pH95) 150mMNaCl and 5mMMgCl

2

27 Activity Assay of Recombinant O furnacalis PPO ProteinsTo check the PO activity of recombinant proteins a conve-nient way was utilized as described by Li et al [30] BrieflyCu2+ was also added into the culture to a final concentra-tion of 05mM upon the addition of IPTG Following therecombinant expression as described above 1mL of E coliculture expressing each OfPPO was centrifuged at 10000 gfor 3min The resulting E coli cells were washed using10mM Tris buffer (pH74) three times and then suspendedin 30 ethanol for nonproteolytic activation of recombinantPPOs After incubation at room temperature for 30min thereaction mixture was centrifuged at 10000 g for 3min Thecell pellet containing PPOs was then incubated with 250120583Lof dopamine (10mM) for 5min Then PO activity of eachsample was measured by monitoring A

490in a microplate

reader (Bio-Tek instrument Inc) One unit of PO activitywasdefined as the amount of enzyme producing an increase inabsorbance (ΔA

490) of 0001 per min

3 Results

31 Cloning and Analysis of O furnacalis PPO cDNAs Withldquonext generationrdquo high-throughput sequencing we obtainedan O furnacalis transcriptome dataset containing 62382unigenes [20] Among these three (1299-bpCL5552Contig12366-bp CL997Contig1 and 1153-bp Unigene28348) werepredicted to encode potential PPOs [20] The 2366-bpCL997Contig1 contains a complete 2079-bp open readingframe and encodes a polypeptide sequencewith 100 identityto O furnacalis PPO (GenBank ABC59699) previouslyreported by Feng et al [37] Here we designated it asOfPPO2We performed 51015840- and 31015840-RACE to obtain the missing endsof the transcript CL5552Contig1 and Unigene28348 Aftervalidation CL5552Contig1 was actually an assembly mosaicconsisting of two variant sequences The obtained full-lengthcDNA was nominated as OfPPO1a OfPPO1b and OfPPO3respectively They were submitted to NCBI and assigned theaccession number as KX452359 KX437622 and KX437621respectively OfPPO1a and OfPPO1b matched with the 31015840-and 51015840-end of CL5552Contig1 respectively (Figure 1(a)) Weconducted PCR using the primers common in bothOfPPO1a


and OfPPO1b (Table S1) and subjected the PCR productsfor sequencing Among 12 clones randomly selected 6 oneswere identical to OfPPO1a while the other 6 were identicaltoOfPPO1b This indicated thatOfPPO1a andOfPPO1bwereexpressed with the same abundance in O furnacalis larvae

The full-length cDNA of OfPPO1a OfPPO1b andOfPPO3 contains 2462 2391 and 2388 nucleotides with a2052- 2058- and 2100-bp open reading frame respectively(Figures 1 and 2) A poly(A) tail was found in the cDNAsequences of OfPPO1b and OfPPO3 but not in OfPPO1aThe conceptual proteins deduced from nucleotide sequencesof OfPPO1a OfPPO1b and OfPPO3 cDNA consist of 683685 and 699 amino acid residues respectively (Figures 1 and2)TheOfPPO2 cDNAencodes a polypeptidewith 692 aminoacid residues [37] All four O furnacalis PPOs including theprevious OfPPO2 lack a typical secretion signal peptide attheir N-terminal regions The calculated molecular mass andisoelectric point of the deduced proteins are 786 kDa and659 for PPO1a 786 kDa and 599 for PPO1b 798 kDa and572 for PPO2 and 810 kDa and 741 for PPO3 respectively

32 Sequence Comparisons and Phylogenetic Analysis of Ofurnacalis PPOs To clarify whether the amino acid residuesand motifs critical for PPO activation and activity were alsoconserved in O furnacalis PPOs the deduced amino acidsequences of four O furnacalis PPOs were aligned withwell-characterized PPOs from B mori [41]M sexta [19 42]and H diomphalia [23 24] The alignments revealed that 6key regions were highly conserved in 4 O furnacalis PPOs(Figure 3) The OfPPOs contain a conserved sequence (Arg-Phe-Gly) around the cleavage and activation site 52RF53 inOfPPO1a 53RF54 in OfPPO1b 51RF52 in OfPPO2 and 52RF53in OfPPO3 (Figure 3(a)) They possibly contain anotherproteolytic cleavage site 164RA165 in OfPPO1a 165KA166 inOfPPO1b 166QA167 in OfPPO2 and 168EA169 in OfPPO3(Figure 3(a)) Two copper binding sites CuA and CuB arevery similar among these proteins 198ndash246 and 358ndash413 inOfPPO1a 199ndash247 and 359ndash414 in OfPPO1b 201ndash250 and358ndash413 in OfPPO2 and 203ndash254 and 362ndash417 in OfPPO3respectively These two copper binding sites carry threeindispensable histidine residues in each site at H210-H214-H239 and H266-H270-H406 in OfPPO1a H211-H215-H240 andH267-H271H407 in OfPPO1b H213-H217-H243 and H266-H270-H406 in OfPPO2 and H215-H219-H247 and H270-H274-H410 inOfPPO3 respectively These conserved histidine residues arelikely to be involved in the copper binding (Figures 3(b) and3(c))Moreover CuAcontains hemocyanin encoding patternslike signature at 209HHWHWHLV216 in OfPPO1a210HHWHWHLV217 in OfPPO1b 212HHWHWHLV219 inOfPPO2 and 214HHWHWHLI221 in OfPPO3 respec-tively CuB contains both hemocyanin signatures(395TAMRDPFFY403 in OfPPO1a 396TAMRDPFFY404 inOfPPO1b 395TTMRDPFFY403 in OfPPO2 and399TTMRDPYFY407 in OfPPO3) and tyrosinase signatures(399DPFFYRWHAYVD410 inOfPPO1a 400DPFFYRWHAFVD411in OfPPO1b 399DPFFYRWHAFID410 in OfPPO2 and403DPYFYRWHAFID414 in OfPPO3) (Figures 3(b) and3(c)) Additionally two conserved motifs are present in the

C-terminal end of all 4 OfPPOs one (CGCGWPHQHML)matches the thiol ester region of 120572-macroglobulin andcomplement proteins C3 and C4 and the other one(MGFPFDR) is nearly identical in all compared PPOs(Figure 3(d)) The catalytic residue (E364) key to thehydroxylation and oxidation activities of Anopheles gambiaePPO8 [43] is identified as E351 in OfPPO1a E352 in OfPPO1bE351 in OfPPO2 E355 in OfPPO3 respectively Besidestwo disulfide bonds are predicted to be in the OfPPOsbased on the comparisons with previously characterized Msexta PPOs [42] and A gambiae PPO8 [43] Cys-581Cys-623 and Cys-583Cys-630 in OfPPO1a Cys-582Cys-624and Cys-584Cys-631 in OfPPO1b Cys-583Cys-627 andCys-585Cys-634 in OfPPO2 and Cys-589Cys-634 andCys-591Cys-641 in OfPPO3 (Figures 1(b) and 2) All aboveresults suggest that the obtained 4 nucleotide acid sequences(OfPPO1a-OfPPO3) encode functional PPO proteins

Database search and sequence alignment indicated that4 O furnacalis PPOs exhibited high amino acid sequencesimilarity (42ndash79 identity) with PPOs from other insectspecies OfPPO1a OfPPO1b OfPPO2 and OfPPO3 aremost similar in amino acid sequences to B mori PPO1(AAG09304) Mythimna separata PPO (BAM76811) Galle-ria mellonella PPO2 (AAQ75026) and G mellonella PPO2(AAQ75026) with the sequence identity of 745 741793 and 705 respectively A phylogenetic analysiswas performed to investigate the evolutionary relationshipsbetween OfPPOs and other homologs As shown in Figure 4insect and crustacean PPOs form separate clades in thephylogenetic tree The aligned insect PPOs (totally 89 PPOsfrom 34 insect species) are clustered into six distinct groupsincluding the subfamilies for two lepidopteran PPOs twodipteran PPOs one coleopteran PPO and one hymenopteranPPO The only inconsistency is Hemiptera PPO (Choris-toneura fumiferana PPO2 ABW16862) is assigned to thegroup of lepidopteran PPOsThese six groups are divided intothree major clades Clade A consists of the conserved PPOsdistributed among various insect orders whereas Clades Band C comprise the paralogs specifically present in Lepi-doptera and Diptera respectively (Figure 4) O furnacalisPPO1a and PPO1b clustered with lepidopteran PPOs in CladeA and OfPPO2 and OfPPO3 grouped with lepidopteranPPOs in Clade B

33 Expression Profiles of 4 PPO Transcripts in O furnacalisWe analyzed the mRNA levels of 4 O furnacalis PPOs invarious development stages different tissues and differentpathogen challenges using semiquantitative RT-PCR meth-ods As shown in Figure 5 the fourO furnacalis PPOs exhib-ited distinct expression patternsTheOfPPO1a transcript wasat the highest levels in eggs and the fifth-instar larval stageand then in the pupae This transcript was barely detectablein the first to fourth-instar larval stage especially in the firstand second-instar larval stage OfPPO1b and OfPPO2 wereexpressed in all tested developmental stages including egglarval and pupal stages However OfPPO1b was expressedat the highest levels in eggs while OfPPO2 was expressed atsimilar levels in all stagesTheOfPPO3mRNA level increasedfrom the second-instar larval stage reached a maximum at


1 AGGGCCTTGGAAATGATTACGCCAGCTTGCATGCCTGCAGGTCGACGATTTGTGAAGTGCAGTATTTTGGTAGGAACTTGAATACAC

TGTTGGTGGAAGATGACGACTTCCGTGGTCAGAGGTCTGGAGCTCCTGTTCGACAGGCCTAACGAGCCCCTGATCACACCGAAAGGC

GAGGAACAAGTCCTGTTCAAACTTAATAAGGAACTGTTGCCCCCGGGCTACGAGGACAACGGCATCGTCCTGAACAACCGCTTCGGCE E Q V L F K L N K E L L P P G Y E D N G I V L N N R F G

GAGGAGCCTGGGGTGCAGACGATCGAGCTGAAGCCGCTGGCGAACCCCCCTCGGTTCAGACGCGCCTCGCAGCTGCCTCGCGATGAA

GACTTCTCGCTCTTCCTGCCGAGGCACCAGGAGATGGCTGATGAGGTCATCGACGTTATTTTAGCGGTGCCTAGAAACCACATGGAA

GAGTTGCTGTCAACTTGCGTGTACGCCAGGGGACGGCTCAACCCGCAGTTGTTCCATTACTGTGCGGCTGTGGCTTTCCTCCACAGA

CATGACACGAAGGCGGTTAAGATCCCGAACTTTGCAGAGACTTTCCCGTCCAAATTTTTAACGTCCACAGTGGTCGGGGAGGCACGA

ACCACTACTTCTGTAATTCCTAAAGGACTGAATCGGCCGATCATAACTATTCCCCGGGACTTCACGGCATCGAACTTGGAGGACGAG

E E P G V Q T I E L K P L A N P P R F R R A S Q L P R D E

D F S L F L P R H Q E M A D E V I D V I L A V P R N H M E

H D T K A V K I P N F A E T F P S K F L T S T V V G E A R

G N R T I V D K D R R G E L F F Y M H Q Q I I A R Y N N E

H R L A Y F R E D L G I N L H H W H W H L I Y P F R A T H

GGCAACCGGACGATCGTCGACAAGGACCGCCGAGGGGAGCTGTTCTTCTATATGCACCAGCAGATCATTGCCAGATACAACAACGAG

CGTCTAAACAACTCTTTGAAGCGCGTTAAGAAGTTCAGTGACTTCCGCGAAGAGATCCCCGAGGCTTACTACCCCAAGCTGGACAGT

CTGACGTCATCGCGCGGGTGGCCGCCGCGGCAGGCCAACATGAAGTGGCAGGACCTGAACCGGCCGGTGAACGGCGTCAACGTCCGC

GTCTCCGATATGGAGAGGTGGCGGAACAATGTACAGGAAGCTATAGCAACTGGGCTCGTTGTCTTGCCGGACAACTCAACGCAGCCG

GACCCGTACTTCTACCGCTGGCACGCGTTCATTGACGACCTCTTCCAGAAGCACAAGGAGTCGCCACACGTTCGCAAGTACCAGAGG

TCTGAGTTGGATTATCCTGGCATCGAGGTCCGTAGCGTAAGCGTCGAGTCGAACACACCGGGAGCCAGGAGCAACGAGCTCAACACC

TTCTGGATGACCAGCGACGTGGACTTGTCGCGCGGACTCGACTTCTCTGACCGCGGGAATGTCTACGTACGGTTCACCCATCTGAAC

CACAGGTCTTTCAGATACGTAATCAACGTGAACAACTCAGGGCGGGCGCGTCGCACCACGGTGCGCATCTTCCTCGCGCCTAAGTTC

AACAACCAGATCATTCGTCAGTCCACCGAGTCTTCCGTGACGATTCCCTTCGAGCAGACGTTCCGTGACCTGTCGCGTACGGGCACC N N Q I I R Q S T E S S V T I P F E Q T F R D L S R T G T

GACCCTGGCGACCCGAACAACCTGGAATTCAACTACTGCGGCTGCGGCTGGCCCCAGCACATGCTGCTGCCAAAGGGTACGGCGGCC

R L N N S L K R V K K F S D F R E E I P E A Y Y P K L D S

88

175

349

436

610

262

1480

1393

958287

1306

1045

871

784

697 CATCGCCTGGCGTACTTCCGTGAGGACTTGGGAATCAACCTGCACCACTGGCACTGGCATTTGATTTACCCGTTCCGCGCCACGCAC

5771828

5191654

1567

5481741

26

55

84

E L L S T C V Y A R G R L N P Q L F H Y C A A V A F L H R

L T S S R G W P P R Q A N M K W Q D L N R P V N G V N V R

258

229

200

T T T S V I P K G L N R P I I T I P R D F T A S N L E D E

523

V S D M E R W R N N V Q E A I A T G L V V L P D N S T Q P

CTCACCATCGACATGCTGGGCAACATGATCGAATCCAGCATCCTGTCGCCCAACCCCGAGCGCTACGGCAGCCTGCACAACAACGGG

1219 CACAGCTTCACCGCCTACATCCACGACCCCACCCACCGCTACCTCGAGTCGCACAGTGTAATGGCGGATGAGGCGACCACCATGCGG

1132L T I D M L G N M I E S S I L S P N P E R Y G S L H N N G

316

H S F T A Y I H D P T H R Y L E S H S V M A D E A T T M R

D P Y F Y R W H A F I D D L F Q K H K E S P H V R K Y Q R

S E L D Y P G I E V R S V S V E S N T P G A R S N E L N T

F W M T S D V D L S R G L D F S D R G N V Y V R F T H L N

374

403

432

461

490

M T T S V V R G L E L L F D R P N E P L I T P K G

H R S F R Y V I N V N N S G R A R R T T V R I F L A P K F

GGCGCCAAATACGTGCTCTTCGTCATGCTGTCCAACTATGAAGGGGATAGGGTGGCGCAAGCTAACGTACATCGCGAGTTGACGTGC

GACGAGGGCGGCCGTGCCTGGCTTCTCTCCGACCAGCGCAAGATGTTCATCGAGATGGACAAGTTTGTTCACGACTTACGACCTGGTD 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

ACCGCCGACCACATCGACGACTTCCTAACCCGCAACATGTTCGTGCAGGACGTCACCATCCGCTTCAGTGACGTCACCGAACAGAACT 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

CCGCGGAACCCGAGGCAACAGTGATCCCCTTTATTTAAACATGGAATAGAATATTTATTCCATGTTGTAATCAGCTTTACCACCTTTP R N P R Q Q

GTGCTTAGCTGAAGTGTTCCGCTTTTGTGTTTTAGAAGACCAGTCTTTTGAATTGTAAAATAATACATACCTAGTAATTTATTTATA

1915 606

2002

2089 664

2176 693

22632350

113

142

345

GAGATATCTTTATTAAATGACCCTTTTGTAAATAACGGTAGCATACAATTTAATTAATAATAACATTATGCAATTGAAAAAAAAAAAA

S S

S S

171

Figure 2 Nucleotide and amino acid sequence of O furnacalis PPO3 The deduced amino acid sequences are shown below the nucleotidesequence of PPO3 (NCBI accession number KX437621) Nucleotides are numbered from the first base at the 51015840-end Amino acid residues werenumbered from the initiatingmethionineThe one-letter code for each amino acid is aligned with the second nucleotide of the correspondingcodon The stop codon is marked with asterisk The cDNA fragment from 31015840-RACE started from the dashed-line arrow to the end Thepredicted proteolytic cleavage bonds are shown in red and marked with red arrow The potential copper binding site A (CuA) copperbinding site B (CuB) the putative thiol ester sites and a conserved motif at the C-terminus are shaded in yellow grey green and purplerespectivelyThe six histidine residues absolutely conserved within the CuA and CuB are in the square boxes Two predicted disulfide linkagesare represented by dashed lines


495048484748494848

596058585758615860

161162160160159163165163165

172173171171170174176174176

LSDRFGTDD--AROfPPO1aOfPPO1b

OfPPO2OfPPO3

BmPPO1

BmPPO2

MsPPO1

MsPPO2

HdPPO1

LSDRFGTDD--AR

VSNRFGEET--KS

LNNRFGDEEEVSR

LSNRFGNEA--TK

LNNRFGDDA--SE

IGNRFGSDA--GR

LNNRFGEEPGVQTLNDRFGDDA--GE

Cleavage sitesVFRRAREVSNVVVFRKAREVSNVV

VFSRAREEATLVVFLRAREVSNVV

VVGEARTTTSVI

VFTQAREAAAVIVFTQARETAAVI

VFSQAREMAAVV

VFRQAREVSSVV

lowast lowastlowastlowastlowastlowast

(a)

OfPPO1aOfPPO1b

OfPPO2OfPPO3

BmPPO1

BmPPO2

MsPPO1

MsPPO2

HdPPO1

lowastlowast lowastlowast lowastlowastlowast lowast lowastlowast lowastlowast lowast lowast lowast

Copper binding site AAYFREDIGINLHHWHWHLVYPFEA---DFAIVNKDRRGELFYYMHQQIIARYAYFREDIGVNLHHWHWHLVYPFET---DFAIVNKDRRGELLYYMHQQIIARY

AYFREDLGINLHHWHWHLVYPFEGA--E-QVVRKDRRGELFYYMHQQVIARY

AYWREDLGINLHHWHWHLVYPFSAS--DEKIVAKDRRGELFFYMHQQIIARY

AYFREDIGINLHHWHWHLVYPFDSA--DRSIVNKDRRGELFYYMHQQIIGRY

AYWREDIGINLHHYHWHLVYPFTAN--DLSIVAKDRRGELFFYMHQQVIARFAYFREDLGINLHHWHWHLIYPFRATHGNRTIVDKDRRGELFFYMHQQIIARY

AYFREDIGINLHHWHWHLVYPFDAA--DRAIVNKDRRGELFYYMHQQIIARY

AYFREDIGVNLHHWHWHLVYPFTAT--DRSIVAKDRRGELFFYMHQQIIARY

lowast lowastlowast lowastlowastlowastlowast lowastlowastlowastlowast lowastlowastlowastlowast lowastlowast

198

199

197

197

196

201

203

201

203

246

247

246

246

244

250

254

250

252

(b)

OfPPO1aOfPPO1b

OfPPO2OfPPO3

BmPPO1

BmPPO2

MsPPO1

MsPPO2

HdPPO1

lowast lowastlowast lowast lowast lowastlowastlowast lowast lowastlowast lowast lowastlowast lowast lowastlowastlowast lowastlowastlowast lowast lowast lowast lowastlowast lowast lowast

Copper binding site B

NRTLYGDLHNMGHVFISYIHDPDHRHLETFGVMGDSATAMRDPVFYRWHAYIDDLF

NRDLYGSIHNNMHSFSAYMHDPEHRYLESFGVIADEATTMRDPFFYRVHAWVDDIFNRELYGSIHNNGHSFTAYMHDPEHRYLEQFGVIADEATTMRDPFFYRWHAYIDDVFNPERYGSLHNNGHSFTAYIHDPTHRYLESHSVMADEATTMRDPYFYRWHAFIDDLFNRDLYGSLHNNGHSFTAYMHDPNHRYLESFGVMADEATTMRDPFFYRWHAFIDDIF

358359358358356358362358360

413414413413411413417413415

NRGYYGDLHNMGHVFCSYAHDPDHRHLEQYGVMGDSATAMRDPFFYRWHAYVDDIF 4NRGYYGDLHNMGHVICSYAHDPDHRHLEQYGVMGDSTTAMRDPFFYRWHAFVDDVF

NRGYYGDLHNMGHVFAAYTHDPDHRHLEQFGVMGDSATAMRDPFFYRWHRFVDDVFNRPYYGDLHNMGHVFISYSHDPDHRHLEQFGVMGDSATAMRDPVFYRWHAYIDDIF

(c)

lowastlowastlowastlowastlowast lowastlowast lowastlowastlowastlowastlowastlowastlowast

581

581580579583589583586

CGCGWPHHMLOfPPO1aOfPPO1b

OfPPO2OfPPO3

BmPPO1

BmPPO2

MsPPO1

MsPPO2

HdPPO1

582590

590589588592598592595

591643

643642641647654647650

644649

649648647653660653656

650CGCGWPHHLL

CGCGWPQHML

CGCGWPQHML

CGCGWPHHML

CGCGWPQHML

CGCGWPHHML

CGCGWPQHMLCGCGWPQHML

MGFPFDRMGFPFDR

MGYPFDR

MGYPFDR

MGYPFDR

MGFPFDR

MGFPFDR

MGFPFDRMGFPFDR

Thiol-ester like motif Conserved motif

(d)

Figure 3 Multiple sequence alignment of conserved regions of insect PPOs Amino acid sequences of PPOs from O furnacalis PPO1a(OfPPO1a KX452359) PPO1b (OfPPO1b KX437622) PPO2 (OfPPO2 ABC59699) PPO3 (OfPPO3 KX437621) B mori PPO1 (BmPPO1AAG09304) PPO2 (BmPPO2 D49371) M sexta PPO1 (MsPPO1 AF003253) PPO2 (MsPPO2 AAC37243) and H diomphalia PPO1(HdPPO1 AB079665) were aligned using the Clustal W Alignments of potential cleavage sites (a) two independent copper binding sites(b and c) and two C-terminal conserved motifs (d) are shown Numbers on both ends of each peptide represented amino acid residuenumbers of the respective proteins Amino acid residues shared by four or more proteins are shown by white letters on black backgroundwhile conservative amino acid substitutions are on grey background Asterisks on the bottom of the alignment denoted amino acids sharedby all proteins while small dots indicate conservatively substituted amino acids The arrows in (a) indicate the potential proteolytic cleavagesites for PPO activation Three large dots in (b) and (c) indicate the histidine residues coordinated with the copper atoms

the fifth-instar stage and decreased dramatically in the pupalstage (Figure 5(a)) RT-PCR analyses showed that the 4 Ofurnacalis PPO mRNAs were all detected in the hemocytesat the highest levels among the tested four tissues includingheads guts hemocytes and fat bodies OfPPO1bmRNA wasnot detected in guts and fat bodies (Figure 5(b))

To check the O furnacalis PPO expression patterns afterexposure to microbial elicitors we analyzed their transcriptlevel after O furnacalis larvae were injected with E coli Mluteus B bassiana or water as a control The result fromRT-PCR assay indicated that all 4 O furnacalis PPO mRNAlevels clearly increased in the larva challenged by bacteria or

fungi especially in the E coli-injected larvae Injection ofMluteus or B bassiana resulted in a much smaller increase intheOfPPO1a transcript level compared to the treatment withE coli (Figure 5(c))

34 Production of Recombinant O furnacalis PPOs in theProkaryotic Expression System As a first step to biochem-ically characterize O furnacalis PPOs we tried to producerecombinant proteins using prokaryotic expression systemAfter several preliminary experiments (data not shown) weselected the pET system for recombinant O furnacalis PPOproduction in which the inserted O furnacalis PPO genes


Tenebrio molitor PPO (AB020738) Tribolium castaneum PPO2 (NP_001034522)

Holotrichia diomphalia PPO1 (AB079665) Tribolium castaneum PPO1 (NP_001034493)

Holotrichia diomphalia PPO2 (AB079664)

Apis mellifera PPO (AAO72539) Nasonia vitripennis PPO1 (NP_001164357) Nasonia vitripennis PPO3 (NP_001164331)

Culex quinquefasciatus PPO6 (XP_001848965) Aedes aegypti PPO6 (XP_001648968) Anopheles darlingi PPO1 (ETN57874) Anopheles gambiae PPO1 (AAC27383)

Drosophila melagaster PPO3 (CG42640) Bactrocera dorsalis PPO (AFP81887)

Drosophila melagaster PPO2 (NP610443) Bactrocera cucurbitae PPO (AFP81886) Musca domestica PPO (AAR84669)

Sarcophaga bullata PPO1 (AF161260) Neobellieria bullata PPO (AAD45526)

Pieris rapae PPO1 (AEA92682) Papilio xuthus PPO1 (ADA60206)

Choristoneura fumiferana PPO1 (ABW16861) Ostrinia furnacalis PPO1a (KX452359)

Ostrinia furnacalis PPO1b (KX437622) Galleria mellonella PPO1 (AAK64363)

Plodia interpunctella PPO (AAU29555) Manduca sexta PPO1 (AF003253)

Bombyx mori PPO1 (AAG09304) Hyphantria cunea PPO1 (AAC34251)

Mythimna separata PPO1 (BAM76811) Spodoptera frugiperda PPO1 (ABB92834)

Ostrinia furnacalis PPO3 (KX437621) Danaus plexippus PPO3 (EHJ77685) Danaus plexippus PPO2 (EHJ77686)

Pieris rapae PPO2 (ADM53096) Papilio xuthus PPO2 (ACS36209)

Papilio xuthus PPO3 (BAM18030) Choristoneura fumiferana PPO2 (ABW16862)

Ostrinia furnacalis PPO2 (ABC59699) Galleria mellonella PPO2 (AAQ75026)

Bombyx mori PPO2 (D49371) Bombyx mandarina PPO2 (ABW06580)

Manduca sexta PPO2 (AAC37243) Hyphantria cunea PPO2 (AAC34256)

Mythimna separata PPO2 (BAM76812) Helicoverpa armigera PPO2 (ABU98653)

Spodoptera exigua PPO (ABS59653) Spodoptera litura PPO2 (AY703825)

Spodoptera frugiperda PPO2 (ABB92835)

Drosophila melagaster PPO1 (CG42639) Sarcophaga bullata PPO2 (AF161261)

Aedes aegypti PPO4 (EAT34239) Armigeres subalbatus PPO4 (AAR88079)Culex quinquefasciatus PPO4 (EDS44938)

Aedes aegypti PPO8 (EAT34240) Anopheles gambiae PPO4 (AJ010193)

Anopheles gambiae PPO5 (AJ010194) Anopheles darlingi PPO5 (ETN65646)

Anopheles gambiae PPO8 (AJ459961) Anopheles gambiae PPO7 (AJ459960)

Aedes aegypti PPO7 (EAT34241) Culex quinquefasciatus PPO7 (EDS44937)

Culex pipiens pallens PPOA3 (ACN78418) Anopheles gambiae PPO9 (CAD31061)

Culex quinquefasciatus PPO5 (EDS44936) Aedes aegypti PPO5 (EAT34242)

Armigeres subalbatus PPO7 (ACZ06576) Anopheles gambiae PPO2 (AF004915) Anopheles darlingi PPO2 (ETN67213)

Aedes aegypti PPO10 (EAT36127) Culex quinquefasciatus PPO8 (EDS34231) Culex quinquefasciatus PPO3 (EDS34230)

Aedes aegypti PPO3 (EAT36126) Armigeres subalbatus PPO3 (AAR88078)


Armigeres subalbatus PPO6 (ABI94392) Anopheles gambiae PPO6 (AJ010195)

Anopheles culicifacies PPO (AA022166) Anopheles stephensi PPO6 (AAC69182)

Anopheles gambiae PPO3 (AF004916) Anopheles darlingi PPO3 (ETN65647)

Culex quinquefasciatus PPO1 (EDS43935) Culex quinquefasciatus PPO2 (EDS44935)

Armigeres subalbatus PPO2 (AF286468) Aedes aegypti PPO1 (AF292114) Armigeres subalbatus PPO1 (AAF70320)

Aedes aegypti PPO2 (AF292113) Armigeres subalbatus PPO5 (ABI94391)

Nebalia kensleyi PPO (ACV33307) Oratosquilla oratoria PPO (ADR50356)

Portunus trituberculatus PPO (ACI46633) Scylla serrata PPO (ABD90511)

Metacarcinus magister PPO (ABB59713) Eriocheir sinensis PPO (ABS19633)

Macrobrachium nipponense PPO (AFA26602) Fenneropenaeus chinensis PPO (ABV60265) Penaeus semisulcatus PPO (AAM77690)

Marsupenaeus japonicus PPO2 (BAB70485) Marsupenaeus japonicus PPO1 (BAB83773)

Litopenaeus vannamei PPO (ABQ45957) Penaeus monodon PPO (ACJ31817)

Procambarus clarkii PPO (ABR12412) Pacifastacus leniusculus PPO1 (CAA58471)

Cherax quadricarinatus PPO (AFN41344) Panulirus longipes PPO2 (ADD70249)

Homarus gammarus PPO (CAE46724) Homarus americanus PPO (AAT73697)

100100

100

86

100

62100

100

100

100

100

100

93100

100

100

100100

100

8285

5453

97

56

99

89

100

100

100100

100

8747

45

43

28

16

100

10070

55

54

38

37

27

20

54

45

97

100

100

100

100100

93

99

92

100

100

72100

100

100100

79

62

100

10099

100

99

7397

98

9178

5738

96

51

23

42

93

93

835248

40

98

99

55

39

95

96

96

98

99100

100

005

Coleopteran PPO

Hymenopteran PPO

Dipteran PPO

Lepidopteran PPO

Lepidopteran PPO

Dipteran PPO

Crustacean PPO

Clade A

Clade B

Clade C

Figure 4 Phylogenetic analysis ofO furnacalis PPOs with other homologsThe used amino acid sequences of 108 PPOs were from 34 insectspecies and 18 crustacean species The names of PPO genes were shown as scientific name of species followed by NCBI accession numberof the gene The O furnacalis PPOs are marked in red The branches specific for coleopteran hymenopteran dipteran lepidopteran andcrustacean PPOs respectively are shaded in squares Numbers at the nodes are bootstrap values as percentage The circled bootstrap valuesindicate that the used insect PPOs were divided into three clades


PPO1a

rpL8

Egg L1 L2 L3 L4 L5 PupaeStage

PPO1b

PPO2

PPO3

(a)

Tissue Head Gut HC FB

PPO1a

rpL8

PPO1b

PPO2

PPO3

(b)

PBS

E co

li

M l

uteu

s

B b

assia

na

Injected

PPO1a

rpL8

PPO1b

PPO2

PPO3

(c)

Figure 5 RT-PCR analysis of the expression of fourO furnacalis PPOs (a) Expression profiles ofOfPPOs at different stages of developmentRNAwas extracted from the whole bodies collected from eggs first-instar (L1) second-instar (L2) third-instar (L3) fourth-instar (L4) fifth-instar (L5) larvae and pupaeThe rpL8 was used as an internal control (b) Expression patterns ofOfPPOs in different tissues ofO furnacalislarvae Tissues including head gut hemocytes (HC) and fat bodies (FB) were collected from day-zero fifth-instar larvae for RNA extractionRT-PCR was performed to assess the transcript level of OfPPOs The rpL8 was used to normalize the templates (c) Expression profiles ofPPOs in O furnacalis larvae upon microbial challenge Day 1 fifth-instar larvae were infected with water E coli M luteus or B bassianaRNA was prepared from the whole bodies 24 h after injection RT-PCR was used to analyze the transcript change of OfPPOs The rpL8 wasused as an internal standard to indicate a consistent total mRNA amount

were under control of strong bacteriophage T7 transcriptionand translation signals and their expression was induced byproviding a source of T7 RNA polymerase in the host BL21(DE3) cells Using a highly simplified and efficient PCR-basedcloning technique [40] the complete open reading frame of 4O furnacalis PPOs was successfully inserted into the pET28avector individually In the constructed plasmids two 6 timesHistags were added into the amino- and carboxyl-terminus ofrecombinant PPO proteins respectively (Figure S1) Addi-tionally we successfully transformed dual plasmids intoBL21 (DE3) cells in order to express pairs of PPOs simul-taneously including OfPPO1a and OfPPO2 OfPPO1a andOfPPO3OfPPO1b andOfPPO2 andOfPPO1b andOfPPO3(Figure S3A)

The recombinant expression of 4 O furnacalis PPOswas evaluated at different temperatures and induction con-ditions to determine the optimum parameters for proteinexpression (data not shown) Eventually three of four Ofurnacalis PPOs (OfPPO1b OfPPO2 and OfPPO3 butnot OfPPO1a) were successfully expressed under the IPTGinduction (Figure 6(a)) Although the insect-produced PPOsare soluble proteins [24 41 42] large proportions of recom-binant OfPPO1b OfPPO2 and OfPPO3 formed insolubleaggregates in the E coli cells even using a low concentration

of IPTG (01mM) and a relatively low temperature (18∘C)These aggregates fractionated with the cell debris followinglysis (Figure 6(a)) Under the same induction and lysis con-ditions the solubility of recombinant OfPPO2 was obviouslyhigher than that of recombinant OfPPO1b and OfPPO3Thesoluble fractions of recombinantOfPPO1b andOfPPO3werenearly undetectable in polyacrylamide gel electrophoresis(Figure 6(a))

Additionally recombinant OfPPO1b OfPPO2 andOfPPO3 but not OfPPO1a could be recognized by antibodyagainst His-tag (Figure 6(b)) The cultured cells were treatedwith 30 ethanol for activating recombinant OfPPOs asdescribed by Li et al [30] The resulting PO activity waschecked using dopamine as substrate As Figure 6(c) shownfour ethanol-treated OfPPOs with the exception of OfPPO1aexhibited detectable PO activities There was no significantdifference among the PO activity of recombinants OfPPO1bOfPPO2 and OfPPO3

4 Discussion

Phenoloxidase (PO) is the important enzyme in the melaninsynthesis a pathway in which some secondary reaction prod-ucts including reactive oxygen species (ROS) and reactive


minus34

minus43

minus55

minus72

minus23

minus95

minus130

kDa1 432minus180

minus34

minus43

minus55

minus72

minus23

minus95

minus130minus180

minus34

minus43

minus55

minus72

minus95

minus130minus180

minus34

minus43

minus55

minus72

minus23

minus95

minus130minus180

(kDa) (kDa) (kDa)1 432 1 432 1 432

OfPPO1a OfPPO1b OfPPO2 OfPPO3

lowastlowast

lowast⋄

(a)

Ant

i-His

1 432 5

minus34

minus43

minus55

minus72

minus95

minus130

minus180(kDa)

(b)

a

b

a

b b

Vector PPO1a PPO1b PPO2 PPO300

03

06

09

12PO

act

ivity

(mO

Dm

in)

(c)

Figure 6 Recombinant expression of four O furnacalis PPOs in E coli (a) SDS-PAGE analysis of recombinant PPOs Four O furnacalisPPOs were expressed in E coli at 18∘C for 12 h The cultured cells were harvested and treated as described in Materials and Methods Theobtained protein samples were then subjected to 10 SDS-PAGE and visualized by Coomassie brilliant blue staining Lane 1 10120583L of celllysate collected before the addition of IPTG Lane 2 5120583L of cell lysate collected 12 h after the addition of IPTG Lane 3 10120583L of supernatantof cell lysate treated by sonication (soluble faction) Lane 4 10120583L of precipitate of cell lysate treated by sonication (insoluble fraction) Thetotal recombinant OfPPO1b OfPPO2 or OfPPO3 is indicated by the asterisk while the insoluble protein is indicated by solid diamondThesoluble OfPPO2 is indicated by hollow diamond (b) Western blot analysis of recombinant PPOsThe obtained samples in (a) were subjectedto immunoblotting using mouse anti-His as primary antibodies Lane 1 1 120583L of cell lysate collected before the addition of IPTG Lanes 2ndash5 1 120583L of cell lysate collected 12 h after the addition of IPTG which contained recombinant OfPPO1a OfPPO1b OfPPO2 and OfPPO3respectively (c) PO activity assay of recombinant PPOs The cell lysate was prepared as described in ldquoMaterials and Methods and incubatedwith 30 ethanol for activation [30] PO activity of each protein sample was measured with dopamine as substrate The bars represent meanplusmn SD (119899 = 3) Bars labeled with different letters (a or b) are significantly different (one-way ANOVA followed by the Newman-Keuls test119901 lt 005)

nitrogen species (RNS) are also generated [3 11] PO plays akey role in multiple physiological processes including woundhealing [44] hemolymph clotting [45] and innate immuneresponse especially in defensive encapsulation and melaniza-tion of foreign organisms [11 46] In this study we clonedfull-length nucleotide sequences for 3 previously unknownOfurnacalis PPOs (OfPPO1a OfPPO1b and OfPPO3) togetherwith OfPPO2 which had been reported previously [37] Weinvestigated the expression profiles of these 4 PPOs andperformed the recombinant expression using the prokaryoticexpression system

The three previously undocumented full-length cDNAsequences for O furnacalis PPOs encode polypeptides withsim700 amino acid residues and apparentmolecular weight ofsim80 kDa As described in other insects [41 47] the conceptualO furnacalis PPO proteins also contain four conservedregions including two possible copper binding sites one thiolester-like motif and one common C-terminal end (Figures1ndash3) The copper binding sites with six absolutely conservedhistidine residues are common in all arthropod POs andother closely related proteins such as arthropod hemocyanins[47] They play an important role in encapsulation of foreign


particles [48] O furnacalis PPOs are potentially responsiblefor oxygen transport and melanization process due to thepresence of these two binding sites The high similarity andthe shared conserved domains of O furnacalis PPOs andarthropod PPOs infer that the identified O furnacalis PPOsin this study are members of the PO family

In addition to the structurally necessary domains andmotifs mentioned above there are other two aspects worthyof note in O furnacalis PPOs Firstly no typical secretionsignal peptide was predicted at the N-terminal region of thededuced O furnacalis PPO1a PPO1b and PPO3 (Figures 1and 2) This is also consistent with the case in O furnacalisPPO2 [37] and other insect PPOs excluding venom PPO inthe parasitoid wasp Pimpla hypochondriaca [11 19 49] InD melanogaster PPO was demonstrated to be released bythe rupture of crystal cells [21] In other arthropods thereare possibly other ways to release PPO than by cell rupture[11 50] So far it is unclear whether and how O furnacalisPPOs are released into the extracellular milieu Secondlythe released PPO exists as zymogen in the hemolymphand must be activated by proteolytic cleavage [11 20] Thepresumed cleavage and activation sites of 4 O furnacalisPPOs were predicted between Arg52 and Phe53 in OfPPO1aArg53 and Phe54 in OfPPO1b Arg51 and Phe52 in OfPPO2and Arg52 and Phe53 in OfPPO3 as previously reported[13 16 46] In H diomphalia HdPPO was cleaved at theconserved bondArg50-Phe51 followed by the second cleavageat Arg162-Ala163 [23 24] In the housefly Musca domesticathe activation of PPO may occur after the cleavage at Arg164-Glu165 [51] The Arg-Ala bond in HdPPO was identified atthe common position in OfPPO1a and OfPPO1b but not inOfPPO2 and OfPPO3 The Arg-Glu bond in MdPPO wasconsistently present inO furnacalis PPOs except for OfPPO3with Arg-Thr instead (Figure 3(a)) We have determinedtwo serine proteases SP13 and SP105 which functioned asprophenoloxidase-activating proteases to cleave and activateOfPPO2 [52] However the precise activation site(s) of Ofurnacalis PPOs is still unknown It is also unclear whetheror not there exist other possible cleavage sites other than thetypical Arg-Phe bond

Insect POs are artificially divided into tyrosinase-typePOs and laccase-type POs in some reports the former canhydroxylate monophenols to o-diphenols (EC 114181) andthen oxidize the o-diphenols to quinones (EC 11031) but itcannot oxidize p-diphenols the latter lack the monophenolmonooxygenase activity but can oxidize both o- and p-diphenols (EC 11032) [53 54] A phylogenetic analysis ofthe 4 O furnacalis PPOs with insect tyrosinase-type andlaccase-type POs revealed two distinct clades with 100bootstrap support All 4 O furnacalis PPOs fell inside thecluster of tyrosinase-type POs (Figure S2) This suggeststhat the identified O furnacalis PPOs are orthologous totyrosinases rather than laccases The precise classificationof O furnacalis PPOs cannot be determined until theiractivities towardmonophenols and p-diphenols are analyzedFurthermore the phylogenetic relationship among O fur-nacalis PPOs and other arthropod tyrosinase-type POs wasproposed in this work Lepidopteran dipteran coleopteran

and hymenopteran PPOs were separated in distinct clustersLepidopteran and dipteran PPOs each fell into two separategroups O furnacalis PPO1a and PPO1b were clustered inone lepidopteran PPO group while PPO2 and PPO3 werein the other lepidopteran PPO group (Figure 4) It suggeststhat lepidopteran PPO genes including O furnacalis PPOsmay have undergone duplication and divergence duringevolution

It is of particular interest that up to 4 O furnacalis PPOsequences are identified in this study since only two PPOgenes have been reported in most lepidopteran species [1316] These 4 O furnacalis PPOs were differentially expressedwith respect to developmental stages tissues or inducedconditions (Figure 5) Even PPO1a and PPO1b which are8682 identical in deduced amino acid sequences alsoexhibited distinct expression patterns For example PPO1amRNAs were detected in all tested tissues including headgut hemocytes and fat bodies while OfPPO1b transcriptswere barely detectable in gut and fat bodies (Figure 5(b))Moreover recombinant OfPPO1b OfPPO2 and OfPPO3could be produced successfully in prokaryotic expressionsystem and be activated by ethanol OfPPO1a failed tobe expressed in E coli (Figure 6) Three PPO genes werealso identified in the swallowtail butterfly Papilio xuthus(Figure 4) [55] Therefore we infer that all identified PPOsindeed exist and potentially perform different functions inOfurnacalis although we currently do not know why O fur-nacalis possesses more PPO genes than other lepidopteransExplanations for the presence of 4 PPO sequences will bepossible only whenmore data on PO function inO furnacalisare obtained Obtaining the recombinant PPO proteins invitro in this study was just the first step to this aim The3 O furnacalis PPOs expressed in E coli were observed tohave obvious enzyme activities after activation by ethanol(Figure 6(c)) In order to potentially increase the solubilitywe also tried to obtain PPO heterodimer by performing thecoexpression but failed possibly due to plasmid instability orincompatibility (Figure S3) In future work we will attemptto produce the recombinant PPOs with higher activity andthen investigate their activation mechanisms and detailedfunctions

Competing Interests

The authors declare that the grant scholarship andor fund-ing mentioned in Acknowledgments did not lead to anycompeting interests Additionally the authors declare thatthere are no competing interests regarding the publication ofthis paper

Authorsrsquo Contributions

S Zhang and F Hong performed the experiments H Songand L Wang helped with the collection of PPO sequencesfrom NCBI S Zhang F Hong and C J An designed theexperiments S Zhang and C J An wrote the paper and QLiu gave critical reviews of the paper Shasha Zhang and FangHong contributed equally to this work


Acknowledgments

The authors thank Dr Kanglai He from the Institute of PlantProtection Chinese Academy of Agricultural Sciences forkindly providing Asian corn borers (O furnacalis (Guenee))eggs This work was supported by the earmarked fund forChina Agriculture Research System (no CARS-29-11) OpenResearch FundProgramof State Key Laboratory of IntegratedPest Management (Chinese IPM1515) and National NaturalScience Foundation of China (31672361)

References

[1] M Hachinohe and H Matsumoto ldquoMechanism of selectivephytotoxicity of L-34-dihydroxyphenylalanine (L-Dopa) inbarnyardglass and lettucerdquo Journal of Chemical Ecology vol 33no 10 pp 1919ndash1926 2007

[2] T Kuraishi A Hori and S Kurata ldquoHost-microbe interactionsin the gut of Drosophila melanogasterrdquo Frontiers in Physiologyvol 4 article no 375 2013

[3] I Kruk K Lichszteld M Bounias A Kladna and L Kubera-Nowakowska ldquoFormation of active oxygen species duringautoxidation of DOPArdquo Chemosphere vol 39 no 3 pp 443ndash453 1999

[4] T Boeckx R Webster A L Winters K J Webb A Gay and AH Kingston-Smith ldquoPolyphenol oxidase-mediated protectionagainst oxidative stress is not associated with enhanced photo-synthetic efficiencyrdquo Annals of Botany vol 116 no 4 pp 529ndash540 2015

[5] E I Solomon U M Sundaram and T E Machonkin ldquoMulti-copper oxidases and oxygenasesrdquoChemical Reviews vol 96 no7 pp 2563ndash2605 1996

[6] F Aguilera C McDougall and B M Degnan ldquoOrigin evo-lution and classification of type-3 copper proteins lineage-specific gene expansions and losses across the Metazoardquo BMCEvolutionary Biology vol 13 no 1 article no 96 2013

[7] K Fujimoto K Masuda N Asada and E Ohnishi ldquoPurifica-tion and characterization of prophenoloxidases from pupae ofDrosophila melanogasterrdquo Journal of Biochemistry vol 113 no3 pp 285ndash291 1993

[8] P-P Bai Y-F Xie G-M Shen D-D Wei and J-J WangldquoPhenoloxidase and its zymogen are required for the larval-pupal transition in Bactrocera dorsalis (Diptera Tephritidae)rdquoJournal of Insect Physiology vol 71 pp 137ndash146 2014

[9] H SMason ldquoOxidasesrdquoAnnual Review of Biochemistry vol 34pp 595ndash634 1965

[10] J P Gillespie M R Kanost and T Trenczek ldquoBiologicalmediators of insect immunityrdquo Annual Review of Entomologyvol 42 pp 611ndash643 1997

[11] L Cerenius B L Lee and K Soderhall ldquoThe proPO-systempros and cons for its role in invertebrate immunityrdquo Trends inImmunology vol 29 no 6 pp 263ndash271 2008

[12] P Zhao J Li Y Wang and H Jiang ldquoBroad-spectrum antimi-crobial activity of the reactive compounds generated in vitroby Manduca sexta phenoloxidaserdquo Insect Biochemistry andMolecular Biology vol 37 no 9 pp 952ndash959 2007

[13] A Lu Q Zhang J Zhang et al ldquoInsect prophenoloxidase theview beyond immunityrdquo Frontiers in Physiology vol 5 articleno 252 2014

[14] R M Waterhouse E V Kriventseva S Meister et al ldquoEvo-lutionary dynamics of immune-related genes and pathways in

disease-vector mosquitoesrdquo Science vol 316 no 5832 pp 1738ndash1743 2007

[15] Z Zou D L Lopez M R Kanost J D Evans and HJiang ldquoComparative analysis of serine protease-related genesin the honey bee genome possible involvement in embryonicdevelopment and innate immunityrdquo Insect Molecular Biologyvol 15 no 5 pp 603ndash614 2006

[16] M Ashida and P T Brey Molecular Mechanisms of ImmuneResponse in Insects Chapman amp Hall London UK 1998

[17] H Tanaka J Ishibashi K Fujita et al ldquoA genome-wide analysisof genes and gene families involved in innate immunity ofBombyx morirdquo Insect Biochemistry and Molecular Biology vol38 no 12 pp 1087ndash1110 2008

[18] T H Kwon S Y Lee J H Lee et al ldquoPurification andcharacterization of prophenoloxidase from the hemolymph ofcoleopteran insect Holotrichia diomphalia larvaerdquo Moleculesand Cells vol 7 no 1 pp 90ndash97 1997

[19] M Hall T Scott M Sugumaran K Soderhall and J H LawldquoProenzyme of Manduca sexta phenol oxidase purificationactivation substrate specificity of the active enzyme andmolec-ular cloningrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 92 no 17 pp 7764ndash7768 1995

[20] Y Liu D X Shen F Zhou G R Wang and C J AnldquoIdentification of immunity-related genes inOstrinia furnacalisagainst entomopathogenic fungi by RNA-seq analysisrdquo PLoSONE vol 9 no 1 Article ID e86436 pp 1ndash24 2014

[21] G Bidla M S Dushay and U Theopold ldquoCrystal cell ruptureafter injury in Drosophila requires the JNK pathway smallGTPases and the TNF homolog eigerrdquo Journal of Cell Sciencevol 120 no 7 pp 1209ndash1215 2007

[22] S Shrestha andY Kim ldquoEicosanoidsmediate prophenoloxidaserelease from oenocytoids in the beet armyworm Spodopteraexiguardquo Insect Biochemistry and Molecular Biology vol 38 no1 pp 99ndash112 2008

[23] S Y Lee T H Kwon J H Hyun et al ldquon vitro activation of pro-phenol-oxidase by two kinds of pro-phenol-oxidase-activatingfactors isolated from hemolymph of coleopteran Holotrichiadiomphalia larvaerdquo European Journal of Biochemistry vol 254no 1 pp 50ndash57 1998

[24] M S Kim M J Baek M H Lee et al ldquoA new easter-type serine protease cleaves a masquerade-like protein duringprophenoloxidase activation in Holotrichia diomphalia larvaerdquoThe Journal of Biological Chemistry vol 277 no 42 pp 39999ndash40004 2002

[25] M J Gorman Y Wang H Jiang and M R Kanost ldquoManducasexta hemolymph proteinase 21 activates prophenoloxidase-activating proteinase 3 in an insect innate immune responseproteinase cascaderdquo Journal of Biological Chemistry vol 282 no16 pp 11742ndash11749 2007

[26] Y Yasuhara Y Koizumi C Katagiri and M Ashida ldquoReex-amination of properties of prophenoloxidase isolated fromlarval hemolymph of the silkworm Bombyx morirdquo Archives ofBiochemistry and Biophysics vol 320 no 1 pp 14ndash23 1995

[27] H Jiang Y Wang X-Q Yu and M R KanostldquoProphenoloxidase-activating proteinase-2 from hemolymphof Manduca sexta A bacteria-inducible serine proteinasecontaining two clip domainsrdquo The Journal of BiologicalChemistry vol 278 no 6 pp 3552ndash3561 2003

[28] H Jiang Y Wang X-Q Yu Y Zhu and M KanostldquoProphenoloxidase-activating proteinase-3 (PAP-3) fromMan-duca sexta hemolymph a clip-domain serine proteinase reg-ulated by serpin-1J and serine proteinase homologsrdquo Insect


Biochemistry and Molecular Biology vol 33 no 10 pp 1049ndash1060 2003

[29] N Asada T Fukumitsu K Fujimoto and K-I Masuda ldquoActi-vation of prophenoloxidase with 2-propanol and other organiccompounds in Drosophila melanogasterrdquo Insect Biochemistryand Molecular Biology vol 23 no 4 pp 515ndash520 1993

[30] X LiMMa F Liu et al ldquoProperties ofDrosophilamelanogasterprophenoloxidases expressed in Escherichia colirdquo Developmen-tal and Comparative Immunology vol 36 no 4 pp 648ndash6562012

[31] D Takahashi B L Garci andM R Kanost ldquoInitiating proteasewithmodular domains interacts with120573-glucan recognition pro-tein to trigger innate immune response in insectsrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 112 no 45 pp 13856ndash13861 2015

[32] C-H Kim S-J Kim H Kan et al ldquoA three-step proteolyticcascade mediates the activation of the peptidoglycan-inducedtoll pathway in an insectrdquo Journal of Biological Chemistry vol283 no 12 pp 7599ndash7607 2008

[33] X-Q Yu H B Jiang Y Wang and M R Kanost ldquoNonprote-olytic serine proteinase homologs are involved in prophenolox-idase activation in the tobacco hornworm Manduca sextardquoInsect Biochemistry andMolecular Biology vol 33 no 2 pp 197ndash208 2003

[34] E De Gregorio S-J Han W-J Lee et al ldquoAn immune-responsive Serpin regulates the melanization cascade inDrosophilardquoDevelopmental Cell vol 3 no 4 pp 581ndash592 2002

[35] M M Afidchao C J M Musters and G R de Snoo ldquoAsiancorn borer (ACB) and non-ACB pests in GM corn (Zea maysL) in the Philippinesrdquo Pest Management Science vol 69 no 7pp 792ndash801 2013

[36] C Feng Q Song W Lu and J Lu ldquoPurification and char-acterization of hemolymph prophenoloxidase from Ostriniafurnacalis (Lepidoptera Pyralidae) larvaerdquo Comparative Bio-chemistry and Physiology Part B Biochemistry and MolecularBiology vol 151 no 2 pp 139ndash146 2008

[37] C Feng J Huang Q Song et al ldquoParasitization by Macrocen-trus cingulum (Hymenoptera Braconidae) influences expres-sion of prophenoloxidase in Asian corn borer Ostrinia fur-nacalisrdquo Archives of Insect Biochemistry and Physiology vol 77no 3 pp 99ndash117 2011

[38] M M Zhang F Zhou Y Chu Z W Zhao and C J AnldquoIdentification and expression profile analysis of antimicro-bial peptideprotein in Asian corn borer Ostrinia furnacalis(Guenee)rdquo International Journal of Biological Sciences vol 9 no9 pp 1004ndash1012 2013

[39] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[40] C Li A Wen B Shen J Lu Y Huang and Y ChangldquoFastCloning a highly simplified purification-free sequence-and ligation-independent PCR cloning methodrdquo BMC Biotech-nology vol 11 article 92 2011

[41] T Kawabata Y Yasuhara M Ochiai S Matsuura and MAshida ldquoMolecular cloning of insect pro-phenol oxidase acopper-containing protein homologous to arthropod hemo-cyaninrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 92 no 17 pp 7774ndash7778 1995

[42] Y C Li Y Wang H B Jiang and J P Deng ldquoCrystal structureof Manduca sexta prophenoloxidase provides insights into the

mechanism of type 3 copper enzymesrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 106 no 40 pp 17002ndash17006 2009

[43] Y Hu Y Wang J Deng and H Jiang ldquoThe structure of aprophenoloxidase (PPO) fromAnopheles gambiae provides newinsights into the mechanism of PPO activationrdquo BMC Biologyvol 14 no 1 article 2 pp 1ndash13 2016

[44] G Bidla THaulingM S Dushay andUTheopold ldquoActivationof insect phenoloxidase after injury endogenous versus foreignelicitorsrdquo Journal of Innate Immunity vol 1 no 4 pp 301ndash3082009

[45] C Karlsson A M Korayem C Scherfer O Loseva M SDushay andUTheopold ldquoProteomic analysis of theDrosophilalarval hemolymph clotrdquoThe Journal of Biological Chemistry vol279 no 50 pp 52033ndash52041 2004

[46] M R Kanost and M J Gorman ldquoPhenoloxidases in insectimmunityrdquo Insect Immunology pp 69ndash96 2008

[47] A P Lourenco M S Zufelato M M Gentile Bitondi and Z LPaulino Simoes ldquoMolecular characterization of a cDNA encod-ing prophenoloxidase and its expression inApismelliferardquo InsectBiochemistry and Molecular Biology vol 35 no 6 pp 541ndash5522005

[48] A L Hughes ldquoEvolution of the arthropod prophenoloxi-dasehexamerin protein familyrdquo Immunogenetics vol 49 no 2pp 106ndash114 1999

[49] N Parkinson I Smith R Weaver and J P Edwards ldquoA newform of arthropod phenoloxidase is abundant in venom of theparasitoid wasp Pimpla hypochondriacardquo Insect Biochemistryand Molecular Biology vol 31 no 1 pp 57ndash63 2001

[50] E Ling and X-Q Yu ldquoProphenoloxidase binds to the surfaceof hemocytes and is involved in hemocyte melanization inManduca sextardquo Insect Biochemistry and Molecular Biology vol35 no 12 pp 1356ndash1366 2005

[51] T Hara T Miyoshi and T Tsukamoto ldquoComparative studieson larval and pupal phenoloxidase of the housefly MuscaDomestica Lrdquo Comparative Biochemistry and Physiology Part BComparative Biochemistry vol 106 no 2 pp 287ndash292 1993

[52] Y Chu Y Liu D Shen F Hong G Wang and C AnldquoSerine proteases SP1 and SP13 mediate the melanizationresponse of Asian corn borer Ostrinia furnacalis againstentomopathogenic fungusBeauveria bassianardquo Journal of Inver-tebrate Pathology vol 128 pp 64ndash72 2015

[53] Y Arakane S Muthukrishnan R W Beeman M R Kanostand K J Kramer ldquoLaccase 2 is the phenoloxidase gene requiredfor beetle cuticle tanningrdquo Proceedings of the National Academyof Sciences of the United States of America vol 102 no 32 pp11337ndash11342 2005

[54] N T Dittmer and M R Kanost ldquoInsect multicopper oxidasesdiversity properties and physiological rolesrdquo Insect Biochem-istry and Molecular Biology vol 40 no 3 pp 179ndash188 2010

[55] R Futahashi H Shirataki T Narita K Mita and H FujiwaraldquoComprehensive microarray-based analysis for stage-specificlarval camouflage pattern-associated genes in the swallowtailbutterfly Papilio xuthusrdquo BMC Biology vol 10 pp 46ndash56 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


M sexta PPO [27 28] and 50Arg-Phe51 followed by thesecond cleavage at 162Arg-Ala163 in H diomphalia PPO [2324] In vitro PPO can also be activated without proteolyticcleavage by certain chemicals such as ethanol or detergents(eg sodium dodecyl sulfate (SDS) cetylpyridinium chloride(CPC)) [29 30]

The activation of PPO is necessary for PO tofunction A model has generally been accepted for PPOactivation in insects upon pathogenic infections orphysical injuries a serine protease cascade is triggeredto activate the prophenoloxidase-activating protease andprophenoloxidase-activating protease then cleaves PPO at aconserved peptide bond to produce PO [11 20] This processinvolves multiple proteins including pattern recognitionproteins [31] a series of serine proteases [32] serine proteasehomologs [33] and serine protease inhibitors [34]

TheAsian corn borerO furnacalis (Gunee) (LepidopteraCrambidae) is an important insect pest in Asia causingserious damage to corn sorghum and millet [35] Howeverknowledge about PPO in O furnacalis is very incompleteFeng et al purified a PPO from O furnacalis hemolymph[36] and cloned its encoding cDNA [37] Our previouswork identified three transcripts for potential PPOs in thetranscriptome of this pest [20] Among these only onedesignated as OfPPO2 contained a complete open read-ing frame [20 37] In this study we cloned a full-lengthnucleotide sequence for another two O furnacalis PPOswhich we calledOfPPO1 andOfPPO3 (GenBank KX437621)OfPPO1 from the transcriptome is actually an assemblymosaic consisting of two variant PPO sequences OfPPO1a(GenBank KX452359) and OfPPO1b (GenBank KX437622)which are 8682 identical in amino acid sequences Weinvestigated the expression profiles of these 4 PPOs indifferent developmental stages in different tissues and uponthe infection of different microorganisms Additionally weperformed the recombinant expression for these 4 proteinsusing a prokaryotic expression system

2 Materials and Methods

21 Insect Rearing O furnacalis larvae were reared on anartificial diet at 28∘Cunder a relative humidity of 70ndash90 anda photoperiod of 16 h light and 8 h darkness [38]

22MolecularCloning ofO furnacalis PPO cDNAs Three tran-scripts for potential PPOs (CL5552contig1 CL997contig1and Unigene28348) were previously identified from O fur-nacalis transcriptome [20] Among these CL997contig1 wasa complete cDNA sequence but another two transcripts(1299-bp CL5552contig1 and 1152-bp Unigene28348) wereincomplete On the basis of these two fragments primers (seeTable S1 in Supplementary Material available at httpdxdoiorg10115520161781803) were designed for 51015840 and 31015840 rapidamplification of cDNA ends (RACE) to obtain the remainingsequences The 51015840 and 31015840 RACE reactions were performedaccording to the manufacturerrsquos instructions for its RACEkit (Takara Biotechnology (Dalian) Co Ltd China) withcDNA from the whole body of fifth-instarO furnacalis larvaecollected 24 h after injection with 3 120583L of Micrococcus luteus

(3 120583g120583L) Some modifications were made to obtain the 31015840-end of CL5552contig1 Briefly after the first-round 31015840 RACEreaction using primer F1 (Table S1) a 1566-bp fragment with225 bp at 51015840-end covered by CL5552contig1 was obtained Itwas then assembled into a 2865-bp cDNA sequence (ldquoPPO1-assembledrdquo in Figure 1(a)) combined with CL5552contig1(Figure 1(a)) However two variant PCR products whichare both inconsistent with expected ldquoPPO1-assembledrdquo wereamplified with selected primer pairs F3R1 or F3R2 (Fig-ure 1(a) Table S1) They were designated as ldquoPPO1a-1strdquo andldquoPPO1b-1strdquo respectively The second-round 51015840 and 31015840 RACEreactions were performed for ldquoPPO1a-1strdquo and ldquoPPO1b-1strdquofollowing the manufacturerrsquos instructions (Figure 1(a)) Theresulting products were cloned and sequenced After assem-bling primers (Table S1) were designed to amplify the full-length cDNA encompassing the entire reading frame usingthe same larval cDNA for RACE The final PCR products(ldquoPPO1ardquo and ldquoPPO1brdquo) were cloned into pMD19-T vector(Takara Biotechnology (Dalian) Co Ltd China) and thenucleotide sequences were confirmed by DNA sequencing

23 Sequence Analysis of O furnacalis PPOs We carriedout a series of sequence analyses for OfPPO1a OfPPO1bOfPPO2 and OfPPO3 The deduced amino acid sequenceswere obtained by using the Translate tool provided by theSwiss Institute Bioinformatics Analysis of deduced aminoacid sequences including prediction of signal peptidemolec-ular weight and isoelectric point was executed in theEXPASY (Expert Protein Analysis System) proteomics server(httpwwwexpasyorg) Characteristic domains or motifswere identified using DNAssist PROSITE profile database(httpprositeexpasyorgscanprosite)

To investigate the phylogenetic relationship betweenO furnacalis PPOs and other arthropod PPOs ldquoprophe-noloxidaserdquo was used as a keyword to search the nonre-dundant database from the National Center for Biotech-nology Information (NCBI httpswwwncbinlmnihgov)The retrieved PPO sequences were aligned with O furnacalisPPO sequences using the Clustal W program Phylogenetictrees were constructed by the neighbor-joining methodusing MEGA Version 5 software [39] For neighbor-joiningmethod gaps were treated as characters and statisticalanalysis was performed using the bootstrap method with1000 replicates

24 Reverse Transcriptase- (RT-) PCR Analysis of the Expres-sion Profiles of O furnacalis PPOs To investigate the changesof O furnacalis PPO transcript levels in several develop-mental stages total RNA samples were individually prepared(119899 = 5) from three different stages including egg larvae andpupa using TRNzol Reagent (TIANGEN Biotech (Beijing)Co Ltd China) 1 120583g of RNA samples equally from 5individual RNA samples in each stage was treated withDNase I (TIANGEN Biotech (Beijing) Co Ltd China)and converted into first-strand cDNA from an oligo (dT)primer following the instructions for QuantScriptRT Kit(TIANGEN Biotech (Beijing) Co Ltd China) The cDNAproducts independently from 3 biological replicates werediluted 10-fold for use as template in RT-PCR experiments



F1-889

RACE(F1)

1300

TAG


PPO1a -1st TAG

Assembled

Assembled

ATG

110ATG TAG

2107

2159

36ATG TAG

2085

PCR (F4R1)

ATG3

TAA2184


TAGATG

ATG TAG129 2184

ATG TAG3 2058

F2-9

F3-77

R1-2414

R2-2517

R3-1101RACE(R3)

R1-2462

F4-75


Assembled

F3-127 R6-2220PCR (F3R6)


PPO1a - 2nd

PPO1a

PPO1b - 1st

PPO1b - 2nd

PPO1b

(1299bp)

(2865bp)

(2410 bp)

(2462bp)

(2383 bp)

(2366bp)

(2094bp)

(2499bp)

126 bp

217 bp

731 bp 412 bp

415 bp231 bp

411 bp

225 bp

884 bp

864bp

1223 bp

1566 bp

1135 bp

98bp

3998400

5998400

5998400 3

998400

(a)


PPO1aPPO1b



S S S S

(b)









600of the






2


490in a microplate



3 Results


































88

175

349

436

610

262

1480

1393

958287

1306

1045

871

784


5771828

5191654

1567

5481741

26

55

84



258

229

200


523





316





374

403

432

461

490











1915 606

2002

2089 664

2176 693

22632350

113

142

345


S S

S S

171



495048484748494848

596058585758615860

161162160160159163165163165

172173171171170174176174176


OfPPO2OfPPO3

BmPPO1

BmPPO2

MsPPO1

MsPPO2

HdPPO1

LSDRFGTDD--AR

VSNRFGEET--KS

LNNRFGDEEEVSR

LSNRFGNEA--TK

LNNRFGDDA--SE

IGNRFGSDA--GR




VVGEARTTTSVI


VFSQAREMAAVV

VFRQAREVSSVV


(a)

OfPPO1aOfPPO1b

OfPPO2OfPPO3

BmPPO1

BmPPO2

MsPPO1

MsPPO2

HdPPO1










198

199

197

197

196

201

203

201

203

246

247

246

246

244

250

254

250

252

(b)

OfPPO1aOfPPO1b

OfPPO2OfPPO3

BmPPO1

BmPPO2

MsPPO1

MsPPO2

HdPPO1





358359358358356358362358360

413414413413411413417413415



(c)


581

581580579583589583586


OfPPO2OfPPO3

BmPPO1

BmPPO2

MsPPO1

MsPPO2

HdPPO1

582590

590589588592598592595

591643

643642641647654647650

644649

649648647653660653656

650CGCGWPHHLL

CGCGWPQHML

CGCGWPQHML

CGCGWPHHML

CGCGWPQHML

CGCGWPHHML


MGFPFDRMGFPFDR

MGYPFDR

MGYPFDR

MGYPFDR

MGFPFDR

MGFPFDR

MGFPFDRMGFPFDR


(d)

























































100100

100

86

100

62100

100

100

100

100

100

93100

100

100

100100

100

8285

5453

97

56

99

89

100

100

100100

100

8747

45

43

28

16

100

10070

55

54

38

37

27

20

54

45

97

100

100

100

100100

93

99

92

100

100

72100

100

100100

79

62

100

10099

100

99

7397

98

9178

5738

96

51

23

42

93

93

835248

40

98

99

55

39

95

96

96

98

99100

100

005

Coleopteran PPO

Hymenopteran PPO

Dipteran PPO

Lepidopteran PPO

Lepidopteran PPO

Dipteran PPO

Crustacean PPO

Clade A

Clade B

Clade C



PPO1a

rpL8


PPO1b

PPO2

PPO3

(a)


PPO1a

rpL8

PPO1b

PPO2

PPO3

(b)

PBS

E co

li

M l

uteu

s

B b

assia

na

Injected

PPO1a

rpL8

PPO1b

PPO2

PPO3

(c)






4 Discussion



minus34

minus43

minus55

minus72

minus23

minus95

minus130

kDa1 432minus180

minus34

minus43

minus55

minus72

minus23

minus95

minus130minus180

minus34

minus43

minus55

minus72

minus95

minus130minus180

minus34

minus43

minus55

minus72

minus23

minus95

minus130minus180

(kDa) (kDa) (kDa)1 432 1 432 1 432


lowastlowast

lowast⋄

(a)

Ant

i-His

1 432 5

minus34

minus43

minus55

minus72

minus95

minus130

minus180(kDa)

(b)

a

b

a

b b


03

06

09

12PO

act

ivity

(mO

Dm

in)

(c)










Competing Interests





Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















F1-889

RACE(F1)

1300

TAG


PPO1a -1st TAG

Assembled

Assembled

ATG

110ATG TAG

2107

2159

36ATG TAG

2085

PCR (F4R1)

ATG3

TAA2184


TAGATG

ATG TAG129 2184

ATG TAG3 2058

F2-9

F3-77

R1-2414

R2-2517

R3-1101RACE(R3)

R1-2462

F4-75


Assembled

F3-127 R6-2220PCR (F3R6)


PPO1a - 2nd

PPO1a

PPO1b - 1st

PPO1b - 2nd

PPO1b

(1299bp)

(2865bp)

(2410 bp)

(2462bp)

(2383 bp)

(2366bp)

(2094bp)

(2499bp)

126 bp

217 bp

731 bp 412 bp

415 bp231 bp

411 bp

225 bp

884 bp

864bp

1223 bp

1566 bp

1135 bp

98bp

3998400

5998400

5998400 3

998400

(a)


PPO1aPPO1b



S S S S

(b)









600of the






2


490in a microplate



3 Results


































88

175

349

436

610

262

1480

1393

958287

1306

1045

871

784


5771828

5191654

1567

5481741

26

55

84



258

229

200


523





316





374

403

432

461

490











1915 606

2002

2089 664

2176 693

22632350

113

142

345


S S

S S

171



495048484748494848

596058585758615860

161162160160159163165163165

172173171171170174176174176


OfPPO2OfPPO3

BmPPO1

BmPPO2

MsPPO1

MsPPO2

HdPPO1

LSDRFGTDD--AR

VSNRFGEET--KS

LNNRFGDEEEVSR

LSNRFGNEA--TK

LNNRFGDDA--SE

IGNRFGSDA--GR




VVGEARTTTSVI


VFSQAREMAAVV

VFRQAREVSSVV


(a)

OfPPO1aOfPPO1b

OfPPO2OfPPO3

BmPPO1

BmPPO2

MsPPO1

MsPPO2

HdPPO1










198

199

197

197

196

201

203

201

203

246

247

246

246

244

250

254

250

252

(b)

OfPPO1aOfPPO1b

OfPPO2OfPPO3

BmPPO1

BmPPO2

MsPPO1

MsPPO2

HdPPO1





358359358358356358362358360

413414413413411413417413415



(c)


581

581580579583589583586


OfPPO2OfPPO3

BmPPO1

BmPPO2

MsPPO1

MsPPO2

HdPPO1

582590

590589588592598592595

591643

643642641647654647650

644649

649648647653660653656

650CGCGWPHHLL

CGCGWPQHML

CGCGWPQHML

CGCGWPHHML

CGCGWPQHML

CGCGWPHHML


MGFPFDRMGFPFDR

MGYPFDR

MGYPFDR

MGYPFDR

MGFPFDR

MGFPFDR

MGFPFDRMGFPFDR


(d)

























































100100

100

86

100

62100

100

100

100

100

100

93100

100

100

100100

100

8285

5453

97

56

99

89

100

100

100100

100

8747

45

43

28

16

100

10070

55

54

38

37

27

20

54

45

97

100

100

100

100100

93

99

92

100

100

72100

100

100100

79

62

100

10099

100

99

7397

98

9178

5738

96

51

23

42

93

93

835248

40

98

99

55

39

95

96

96

98

99100

100

005

Coleopteran PPO

Hymenopteran PPO

Dipteran PPO

Lepidopteran PPO

Lepidopteran PPO

Dipteran PPO

Crustacean PPO

Clade A

Clade B

Clade C



PPO1a

rpL8


PPO1b

PPO2

PPO3

(a)


PPO1a

rpL8

PPO1b

PPO2

PPO3

(b)

PBS

E co

li

M l

uteu

s

B b

assia

na

Injected

PPO1a

rpL8

PPO1b

PPO2

PPO3

(c)






4 Discussion



minus34

minus43

minus55

minus72

minus23

minus95

minus130

kDa1 432minus180

minus34

minus43

minus55

minus72

minus23

minus95

minus130minus180

minus34

minus43

minus55

minus72

minus95

minus130minus180

minus34

minus43

minus55

minus72

minus23

minus95

minus130minus180

(kDa) (kDa) (kDa)1 432 1 432 1 432


lowastlowast

lowast⋄

(a)

Ant

i-His

1 432 5

minus34

minus43

minus55

minus72

minus95

minus130

minus180(kDa)

(b)

a

b

a

b b


03

06

09

12PO

act

ivity

(mO

Dm

in)

(c)










Competing Interests





Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















600of the






2


490in a microplate



3 Results


































88

175

349

436

610

262

1480

1393

958287

1306

1045

871

784


5771828

5191654

1567

5481741

26

55

84



258

229

200


523





316





374

403

432

461

490











1915 606

2002

2089 664

2176 693

22632350

113

142

345


S S

S S

171



495048484748494848

596058585758615860

161162160160159163165163165

172173171171170174176174176


OfPPO2OfPPO3

BmPPO1

BmPPO2

MsPPO1

MsPPO2

HdPPO1

LSDRFGTDD--AR

VSNRFGEET--KS

LNNRFGDEEEVSR

LSNRFGNEA--TK

LNNRFGDDA--SE

IGNRFGSDA--GR




VVGEARTTTSVI


VFSQAREMAAVV

VFRQAREVSSVV


(a)

OfPPO1aOfPPO1b

OfPPO2OfPPO3

BmPPO1

BmPPO2

MsPPO1

MsPPO2

HdPPO1










198

199

197

197

196

201

203

201

203

246

247

246

246

244

250

254

250

252

(b)

OfPPO1aOfPPO1b

OfPPO2OfPPO3

BmPPO1

BmPPO2

MsPPO1

MsPPO2

HdPPO1





358359358358356358362358360

413414413413411413417413415



(c)


581

581580579583589583586


OfPPO2OfPPO3

BmPPO1

BmPPO2

MsPPO1

MsPPO2

HdPPO1

582590

590589588592598592595

591643

643642641647654647650

644649

649648647653660653656

650CGCGWPHHLL

CGCGWPQHML

CGCGWPQHML

CGCGWPHHML

CGCGWPQHML

CGCGWPHHML


MGFPFDRMGFPFDR

MGYPFDR

MGYPFDR

MGYPFDR

MGFPFDR

MGFPFDR

MGFPFDRMGFPFDR


(d)

























































100100

100

86

100

62100

100

100

100

100

100

93100

100

100

100100

100

8285

5453

97

56

99

89

100

100

100100

100

8747

45

43

28

16

100

10070

55

54

38

37

27

20

54

45

97

100

100

100

100100

93

99

92

100

100

72100

100

100100

79

62

100

10099

100

99

7397

98

9178

5738

96

51

23

42

93

93

835248

40

98

99

55

39

95

96

96

98

99100

100

005

Coleopteran PPO

Hymenopteran PPO

Dipteran PPO

Lepidopteran PPO

Lepidopteran PPO

Dipteran PPO

Crustacean PPO

Clade A

Clade B

Clade C



PPO1a

rpL8


PPO1b

PPO2

PPO3

(a)


PPO1a

rpL8

PPO1b

PPO2

PPO3

(b)

PBS

E co

li

M l

uteu

s

B b

assia

na

Injected

PPO1a

rpL8

PPO1b

PPO2

PPO3

(c)






4 Discussion



minus34

minus43

minus55

minus72

minus23

minus95

minus130

kDa1 432minus180

minus34

minus43

minus55

minus72

minus23

minus95

minus130minus180

minus34

minus43

minus55

minus72

minus95

minus130minus180

minus34

minus43

minus55

minus72

minus23

minus95

minus130minus180

(kDa) (kDa) (kDa)1 432 1 432 1 432


lowastlowast

lowast⋄

(a)

Ant

i-His

1 432 5

minus34

minus43

minus55

minus72

minus95

minus130

minus180(kDa)

(b)

a

b

a

b b


03

06

09

12PO

act

ivity

(mO

Dm

in)

(c)










Competing Interests





Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















































88

175

349

436

610

262

1480

1393

958287

1306

1045

871

784


5771828

5191654

1567

5481741

26

55

84



258

229

200


523





316





374

403

432

461

490











1915 606

2002

2089 664

2176 693

22632350

113

142

345


S S

S S

171



495048484748494848

596058585758615860

161162160160159163165163165

172173171171170174176174176


OfPPO2OfPPO3

BmPPO1

BmPPO2

MsPPO1

MsPPO2

HdPPO1

LSDRFGTDD--AR

VSNRFGEET--KS

LNNRFGDEEEVSR

LSNRFGNEA--TK

LNNRFGDDA--SE

IGNRFGSDA--GR




VVGEARTTTSVI


VFSQAREMAAVV

VFRQAREVSSVV


(a)

OfPPO1aOfPPO1b

OfPPO2OfPPO3

BmPPO1

BmPPO2

MsPPO1

MsPPO2

HdPPO1










198

199

197

197

196

201

203

201

203

246

247

246

246

244

250

254

250

252

(b)

OfPPO1aOfPPO1b

OfPPO2OfPPO3

BmPPO1

BmPPO2

MsPPO1

MsPPO2

HdPPO1





358359358358356358362358360

413414413413411413417413415



(c)


581

581580579583589583586


OfPPO2OfPPO3

BmPPO1

BmPPO2

MsPPO1

MsPPO2

HdPPO1

582590

590589588592598592595

591643

643642641647654647650

644649

649648647653660653656

650CGCGWPHHLL

CGCGWPQHML

CGCGWPQHML

CGCGWPHHML

CGCGWPQHML

CGCGWPHHML


MGFPFDRMGFPFDR

MGYPFDR

MGYPFDR

MGYPFDR

MGFPFDR

MGFPFDR

MGFPFDRMGFPFDR


(d)

























































100100

100

86

100

62100

100

100

100

100

100

93100

100

100

100100

100

8285

5453

97

56

99

89

100

100

100100

100

8747

45

43

28

16

100

10070

55

54

38

37

27

20

54

45

97

100

100

100

100100

93

99

92

100

100

72100

100

100100

79

62

100

10099

100

99

7397

98

9178

5738

96

51

23

42

93

93

835248

40

98

99

55

39

95

96

96

98

99100

100

005

Coleopteran PPO

Hymenopteran PPO

Dipteran PPO

Lepidopteran PPO

Lepidopteran PPO

Dipteran PPO

Crustacean PPO

Clade A

Clade B

Clade C



PPO1a

rpL8


PPO1b

PPO2

PPO3

(a)


PPO1a

rpL8

PPO1b

PPO2

PPO3

(b)

PBS

E co

li

M l

uteu

s

B b

assia

na

Injected

PPO1a

rpL8

PPO1b

PPO2

PPO3

(c)






4 Discussion



minus34

minus43

minus55

minus72

minus23

minus95

minus130

kDa1 432minus180

minus34

minus43

minus55

minus72

minus23

minus95

minus130minus180

minus34

minus43

minus55

minus72

minus95

minus130minus180

minus34

minus43

minus55

minus72

minus23

minus95

minus130minus180

(kDa) (kDa) (kDa)1 432 1 432 1 432


lowastlowast

lowast⋄

(a)

Ant

i-His

1 432 5

minus34

minus43

minus55

minus72

minus95

minus130

minus180(kDa)

(b)

a

b

a

b b


03

06

09

12PO

act

ivity

(mO

Dm

in)

(c)










Competing Interests





Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















495048484748494848

596058585758615860

161162160160159163165163165

172173171171170174176174176


OfPPO2OfPPO3

BmPPO1

BmPPO2

MsPPO1

MsPPO2

HdPPO1

LSDRFGTDD--AR

VSNRFGEET--KS

LNNRFGDEEEVSR

LSNRFGNEA--TK

LNNRFGDDA--SE

IGNRFGSDA--GR




VVGEARTTTSVI


VFSQAREMAAVV

VFRQAREVSSVV


(a)

OfPPO1aOfPPO1b

OfPPO2OfPPO3

BmPPO1

BmPPO2

MsPPO1

MsPPO2

HdPPO1










198

199

197

197

196

201

203

201

203

246

247

246

246

244

250

254

250

252

(b)

OfPPO1aOfPPO1b

OfPPO2OfPPO3

BmPPO1

BmPPO2

MsPPO1

MsPPO2

HdPPO1





358359358358356358362358360

413414413413411413417413415



(c)


581

581580579583589583586


OfPPO2OfPPO3

BmPPO1

BmPPO2

MsPPO1

MsPPO2

HdPPO1

582590

590589588592598592595

591643

643642641647654647650

644649

649648647653660653656

650CGCGWPHHLL

CGCGWPQHML

CGCGWPQHML

CGCGWPHHML

CGCGWPQHML

CGCGWPHHML


MGFPFDRMGFPFDR

MGYPFDR

MGYPFDR

MGYPFDR

MGFPFDR

MGFPFDR

MGFPFDRMGFPFDR


(d)

























































100100

100

86

100

62100

100

100

100

100

100

93100

100

100

100100

100

8285

5453

97

56

99

89

100

100

100100

100

8747

45

43

28

16

100

10070

55

54

38

37

27

20

54

45

97

100

100

100

100100

93

99

92

100

100

72100

100

100100

79

62

100

10099

100

99

7397

98

9178

5738

96

51

23

42

93

93

835248

40

98

99

55

39

95

96

96

98

99100

100

005

Coleopteran PPO

Hymenopteran PPO

Dipteran PPO

Lepidopteran PPO

Lepidopteran PPO

Dipteran PPO

Crustacean PPO

Clade A

Clade B

Clade C



PPO1a

rpL8


PPO1b

PPO2

PPO3

(a)


PPO1a

rpL8

PPO1b

PPO2

PPO3

(b)

PBS

E co

li

M l

uteu

s

B b

assia

na

Injected

PPO1a

rpL8

PPO1b

PPO2

PPO3

(c)






4 Discussion



minus34

minus43

minus55

minus72

minus23

minus95

minus130

kDa1 432minus180

minus34

minus43

minus55

minus72

minus23

minus95

minus130minus180

minus34

minus43

minus55

minus72

minus95

minus130minus180

minus34

minus43

minus55

minus72

minus23

minus95

minus130minus180

(kDa) (kDa) (kDa)1 432 1 432 1 432


lowastlowast

lowast⋄

(a)

Ant

i-His

1 432 5

minus34

minus43

minus55

minus72

minus95

minus130

minus180(kDa)

(b)

a

b

a

b b


03

06

09

12PO

act

ivity

(mO

Dm

in)

(c)










Competing Interests





Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



































































100100

100

86

100

62100

100

100

100

100

100

93100

100

100

100100

100

8285

5453

97

56

99

89

100

100

100100

100

8747

45

43

28

16

100

10070

55

54

38

37

27

20

54

45

97

100

100

100

100100

93

99

92

100

100

72100

100

100100

79

62

100

10099

100

99

7397

98

9178

5738

96

51

23

42

93

93

835248

40

98

99

55

39

95

96

96

98

99100

100

005

Coleopteran PPO

Hymenopteran PPO

Dipteran PPO

Lepidopteran PPO

Lepidopteran PPO

Dipteran PPO

Crustacean PPO

Clade A

Clade B

Clade C



PPO1a

rpL8


PPO1b

PPO2

PPO3

(a)


PPO1a

rpL8

PPO1b

PPO2

PPO3

(b)

PBS

E co

li

M l

uteu

s

B b

assia

na

Injected

PPO1a

rpL8

PPO1b

PPO2

PPO3

(c)






4 Discussion



minus34

minus43

minus55

minus72

minus23

minus95

minus130

kDa1 432minus180

minus34

minus43

minus55

minus72

minus23

minus95

minus130minus180

minus34

minus43

minus55

minus72

minus95

minus130minus180

minus34

minus43

minus55

minus72

minus23

minus95

minus130minus180

(kDa) (kDa) (kDa)1 432 1 432 1 432


lowastlowast

lowast⋄

(a)

Ant

i-His

1 432 5

minus34

minus43

minus55

minus72

minus95

minus130

minus180(kDa)

(b)

a

b

a

b b


03

06

09

12PO

act

ivity

(mO

Dm

in)

(c)










Competing Interests





Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















PPO1a

rpL8


PPO1b

PPO2

PPO3

(a)


PPO1a

rpL8

PPO1b

PPO2

PPO3

(b)

PBS

E co

li

M l

uteu

s

B b

assia

na

Injected

PPO1a

rpL8

PPO1b

PPO2

PPO3

(c)






4 Discussion



minus34

minus43

minus55

minus72

minus23

minus95

minus130

kDa1 432minus180

minus34

minus43

minus55

minus72

minus23

minus95

minus130minus180

minus34

minus43

minus55

minus72

minus95

minus130minus180

minus34

minus43

minus55

minus72

minus23

minus95

minus130minus180

(kDa) (kDa) (kDa)1 432 1 432 1 432


lowastlowast

lowast⋄

(a)

Ant

i-His

1 432 5

minus34

minus43

minus55

minus72

minus95

minus130

minus180(kDa)

(b)

a

b

a

b b


03

06

09

12PO

act

ivity

(mO

Dm

in)

(c)










Competing Interests





Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















minus34

minus43

minus55

minus72

minus23

minus95

minus130

kDa1 432minus180

minus34

minus43

minus55

minus72

minus23

minus95

minus130minus180

minus34

minus43

minus55

minus72

minus95

minus130minus180

minus34

minus43

minus55

minus72

minus23

minus95

minus130minus180

(kDa) (kDa) (kDa)1 432 1 432 1 432


lowastlowast

lowast⋄

(a)

Ant

i-His

1 432 5

minus34

minus43

minus55

minus72

minus95

minus130

minus180(kDa)

(b)

a

b

a

b b


03

06

09

12PO

act

ivity

(mO

Dm

in)

(c)










Competing Interests





Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















Competing Interests





Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Cloning, Expression, and Characterization...

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Transcript of Research Article Cloning, Expression, and Characterization...