[Recombinant expression of black sesame polyphenol oxidase and its enzymatic properties].

To investigate the enzyme properties of the black sesame polyphenol oxidase (BsPPO), a synthesized Bsppo gene was cloned into the vector pMAL-c5x and expressed in E. coli. Subsequently, the MBP fusion label in the recombinant protein was removed by protease digestion after affinity purification. The synthesized Bsppo gene contained 1 752 bp which encodes 585 amino acids with a deduced molecular weight of 65.3 kDa. Transformation of the recombinant vector into E. coli BL21(DE3) resulted in soluble expression of the fusion protein MBP-BsPPO. The enzymatic properties of the recombinant BsPPO was investigated after MBP fusion tag excision followed by affinity purification. The results demonstrated that the optimal temperature and pH for BsPPO was 25°C and 4.0, respectively. BsPPO exhibited a good stability under low temperature and acidic environment. Low-intensity short-term light exposure increased the activity of BsPPO. Cu²âº could improve the activity of BsPPO while Zn²âº and Ca²âº showed the opposite effect. BsPPO could catalyze the oxidation of monophenols, diphenols, and triphenols, and exhibited good catalytic activity on l-tyrosine and vanillic acid. Moreover, BsPPO exhibited high catalytic activity on black sesame metabolites, including 2-methoxy cinnamic acid, indole-3-carboxylic acid and phloretin. These results may serve as a basis for further characterization of BsPPO.

An approach to recombinantly produce mature grape polyphenol oxidase.

Polyphenol oxidases (PPOs) are important enzymes that are widely found in both prokaryotes and eukaryotes including grapes. Studies of grape PPO to date have mostly relied on enzymes extracted and purified from plants. In this work, we describe the production of the mature form of Shine Muscat grape PPO by using an Escherichia coli expression system. We have optimised the purification procedure to obtain pure and active recombinant enzymes and characterised the catalytic efficiency of the recombinant grape PPO by using ultraviolet/visible (UV/Vis) spectrophotometry. Our work provides a simple protocol of obtaining pure and active recombinant grape PPO that will enable further studies about the catalytic mechanism and inhibition of this enzyme.

Enzymatic gene expression by Pleurotus tuoliensis (Bailinggu): differential regulation under low temperature induction conditions.

Pleurotus tuoliensis is a valuable, rare and edible mushroom that is been commercially cultivated and is rapidly developing in China markets. Low temperatures are required to induces primordia initiation for the successful production of fruiting bodies (basidiomes) during commercial cultivation. In this work, we investigated the enzymatic activities and performed transcription profiling analysis of enzymatic genes under different low temperature conditions. The results suggest that the enzymatic activities and transcription levels decrease or increase significantly at 4 and 13 °C. Lacc10 and mnp6 seems to play a dominant role during nutrition growth. Furthermore, the expression of laccase and peroxidase genes was highly correlated to the detected extracellular enzymatic activity. Cold stress genes expression profiles were upregulated under 4 °C/13 °C (3 days), while only the Hsp70 gene was downregulated (at the stage of fruiting bodies production) at 13 °C (12 days). Our results showed that the transcriptional regulation of laccase and ligninolytic peroxidase genes plays an important role in the fruiting bodies of Bailinggu under low temperature induction (4 °C). Induction at low temperatures was a highly important cultivation condition in Bailinggu.

Varroa destructor parasitism reduces hemocyte concentrations and prophenol oxidase gene expression in bees from two populations.

Circulating hemocytes are responsible for defensive and healing mechanisms in the honey bee, Apis mellifera. Parasitism by the mite Varroa destructor and injection of V. destructor homogenate in buffer, but not buffer injection, showed similar reductions in total hemocyte concentrations in both Africanized and European adult honey bees. This indicated that compounds in V. destructor homogenate can have similar effects as V. destructor parasitism and that the response is not solely due to wounding. Samples from honey bees with different hemocyte concentrations were compared for the expression patterns of hemolectin (AmHml), prophenol oxidase (AmPpo), and class C scavenger receptor (AmSRC-C). Of the genes tested, only the expression of AmPpo correlated well with hemocyte counts for all the treatments, indicating that melanization is associated with those responses. Thus, the expression of AmPpo might be a suitable biomarker for hemocyte counts as part of cellular defenses against injection of buffer or mite compounds and V. destructor parasitism and perhaps other conditions involving healing and immunity.

Bacillus spp., a bio-control agent enhances the activity of antioxidant defense enzymes in rice against Pyricularia oryzae.

Plant growth promoting rhizobacteria (PGPR) are found to control the plant diseases by adopting various mechanisms. Induced systemic resistance (ISR) is an important defensive strategy manifested by plants against numerous pathogens especially infecting at aerial parts. Rhizobacteria elicit ISR by inducing different pathways in plants through production of various metabolites. In the present study, potential of Bacillus spp. KFP-5, KFP-7, KFP-17 was assessed to induce antioxidant enzymes against Pyricularia oryzae infection in rice. The antagonistic Bacillus spp. significantly induced antioxidant defense enzymes i-e superoxide dismutase (1.7-1.9-fold), peroxidase (3.5-4.1-fold), polyphenol oxidase (3.0-3.8-fold), phenylalanine ammonia-lyase (3.9-4.4-fold), in rice leaves and roots under hydroponic and soil conditions respectively. Furthermore, the antagonistic Bacillus spp significantly colonized the rice plants (2.0E+00-9.1E+08) and secreted multiple biocontrol determinants like protease (1.1-5.5 U/mg of soil or U/mL of hydroponic solution), glucanase, (1.0-1.3 U/mg of soil or U/mL of hydroponic solution), siderophores (6.5-42.8 µg/mL or mg) in the rhizosphere of different rice varieties. The results showed that treatment with Bacillus spp. enhanced the antioxidant defense activities in infected rice, thus alleviating P. oryzae induced oxidative damage and suppressing blast disease incidence.

Characterization and expression analysis of the prophenoloxidase activating factor from the mud crab Scylla paramamosain.

Prophenoloxidase activating factors (PPAFs) are a group of clip domain serine proteinases that can convert prophenoloxidase (pro-PO) to the active form of phenoloxidase (PO), causing melanization of pathogens. Here, two full-length PPAF cDNAs from Scylla paramamosain (SpPPAF1 and SpPPAF2) were cloned and characterized. The full-length SpPPAF1 cDNA was 1677 bp in length, including a 5'-untranslated region (UTR) of 52 bp, an open reading frame (ORF) of 1131 bp coding for a polypeptide of 376 amino acids, and a 3'-UTR of 494 bp. The full-length SpPPAF2 cDNA was 1808 bp in length, including a 5'-UTR of 88 bp, an ORF of 1125 bp coding for a polypeptide of 374 amino acids, and a 3'-UTR of 595 bp. The estimated molecular weight of SpPPAF1 and SpPPAF2 was 38.43 and 38.56 kDa with an isoelectric point of 7.54 and 7.14, respectively. Both SpPPAF1 and SpPPAF2 proteins consisted of a signal peptide, a characteristic structure of clip domain, and a carboxyl-terminal trypsin-like serine protease domain. Expression analysis by qRT-PCR showed that SpPPAF1 mRNA was mainly expressed in the gill, testis, and hemocytes, and SpPPAF2 mRNA was mainly expressed in hemocytes. In addition, SpPPAF1 and SpPPAF2 mRNA was expressed in a time-dependent manner after Vibrio parahaemolyticus challenge. The results showed that expression of both SpPPAF1 and SpPPAF2 was related to the bacterial challenge but the expression patterns differed. These findings suggest that SpPPAF is a serine proteinase and may be involved in the pro-PO activation pathway of the crab innate immune system.

A novel Lozenge gene in silkworm, Bombyx mori regulates the melanization response of hemolymph.

Runt-related (RUNX) transcription factors are evolutionarily conserved either in vertebrate or invertebrate. Lozenge (Lz), a members of RUNX family as well as homologue of AML-1, functions as an important transcription factor regulating the hemocytes differentiation. In this paper, we identified and characterized RUNX family especially Lz in silkworm, which is a lepidopteran model insect. The gene expression analysis illustrated that BmLz was highly expressed in hemocytes throughout the whole development period, and reached a peak in glutonous stage. Over-expression of BmLz in silkworm accelerated the melanization process of hemolymph, and led to instantaneously up-regulation of prophenoloxidases (PPOs), which were key enzymes in the melanization process. Further down-regulation of BmLz expression by RNA interference resulted in the significant delay of melanization reaction of hemolymph. These findings suggested that BmLz regulated the melanization process of hemolymph by inducing PPOs expression, and played a critical role in innate immunity defense in silkworm.

Cloning and functional expression in E. coli of a polyphenol oxidase transcript from Coreopsis grandiflora involved in aurone formation.

Polyphenol oxidases are involved in aurone biosynthesis but the gene responsible for 4-deoxyaurone formation in Asteraceae was so far unknown. Three novel full-length cDNA sequences were isolated from Coreopsis grandiflora with sizes of 1.80kb (cgAUS1) and 1.85kb (cgAUS2a, 2b), encoding for proteins of 68-69kDa, respectively. cgAUS1 is preferably expressed in young petals indicating a specific role in pigment formation. The 58.9kDa AUS1 holoproenzyme, was recombinantly expressed in E. coli and purified to homogeneity. The enzyme shows only diphenolase activity, catalyzing the conversion of chalcones to aurones and was characterized by SDS-PAGE and shot-gun type nanoUHPLC-ESI-MS/MS.

Cloning and expression analysis of litchi (Litchi Chinensis Sonn.) polyphenol oxidase gene and relationship with postharvest pericarp browning.

Polyphenol oxidase (PPO) plays a key role in the postharvest pericarp browning of litchi fruit, but its underlying mechanism remains unclear. In this study, we cloned the litchi PPO gene (LcPPO, JF926153), and described its expression patterns. The LcPPO cDNA sequence was 2120 bps in length with an open reading frame (ORF) of 1800 bps. The ORF encoded a polypeptide with 599 amino acid residues, sharing high similarities with other plant PPO. The DNA sequence of the ORF contained a 215-bp intron. After carrying out quantitative RT-PCR, we proved that the LcPPO expression was tissue-specific, exhibiting the highest level in the flower and leaf. In the pericarp of newly-harvested litchi fruits, the LcPPO expression level was relatively high compared with developing fruits. Regardless of the litchi cultivar and treatment conditions, the LcPPO expression level and the PPO activity in pericarp of postharvest fruits exhibited the similar variations. When the fruits were stored at room temperature without packaging, all the pericarp browning index, PPO activity and the LcPPO expression level of litchi pericarps were reaching the highest in Nandaowuhe (the most rapid browning cultivar), but the lowest in Ziniangxi (the slowest browning cultivar) within 2 d postharvest. Preserving the fruits of Feizixiao in 0.2-µm plastic bag at room temperature would decrease the rate of pericarp water loss, delay the pericarp browning, and also cause the reduction of the pericarp PPO activity and LcPPO expression level within 3 d postharvest. In addition, postharvest storage of Feizixiao fruit stored at 4°C delayed the pericarp browning while decreasing the pericarp PPO activity and LcPPO expression level within 2 d after harvest. Thus, we concluded that the up-regulation of LcPPO expression in pericarp at early stage of postharvest storage likely enhanced the PPO activity and further accelerated the postharvest pericarp browning of litchi fruit.

Activity of fusion prophenoloxidase-GFP and its potential applications for innate immunity study.

Insect prophenoloxidase (PPO) is essential for physiological functions such as melanization of invading pathogens, wound healing and cuticle sclerotization. The insect PPO activation pathway is well understood. However, it is not very clear how PPO is released from hemocytes and how PPO takes part in cellular immunity. To begin to assess this, three Drosophila melanogaster PPO genes were separately fused with GFP at the C-terminus (rPPO-GFP) and were over-expressed in S2 cells. The results of staining and morphological observation show that rPPO-GFP expressed in S2 cells has green fluorescence and enzyme activity if Cu(2+) was added during transfection. Each rPPO-GFP has similar properties as the corresponding rPPO. However, cells with rPPO-GFP over-expressed are easier to trace without PO activation and staining. Further experiments show that rPPO1-GFP is cleaved and activated by Drosophila serine protease, and rPPO1-GFP binds to Micrococcus luteus and Beauveria bassiana spores as silkworm plasma PPO. The above research indicates that the GFP-tag has no influence on the fusion enzyme activation and PPO-involved innate immunity action in vitro. Thus, rPPO-GFP may be a convenient tool for innate immunity study in the future if it can be expressed in vivo.

Cytological and physiological basis for tomato varietal resistance against Alternaria alternata.

BACKGROUND: Since tomato is an important food component, it is imperative to enhance its yield against the activities of many devastating fungal pathogens such as Alternaria alternata. The exploitation of plant innate resistance by cultivation of resistant varieties is an effective measure in this regard. In the present study, 28 tomato varieties were tested against 32 A. alternata isolates, and representative varieties were further evaluated to determine the extent and basis of their antifungal resistance. RESULTS: A significant increase (104.7%) in polyphenols was recorded in the resistant variety Dinaar compared with the susceptible variety Red Tara. Dinaar also exhibited 100% enhancement of alkaloids and terpenoids along with a 30.7% increase in cell wall hemicellulose content. Significant differences were found in physical barriers (cellulose, lignin and pectin) of the representative varieties when stained tissue sections were subjected to colorimetric analysis. Similarly, polyphenol oxidase, peroxidase and phenylalanine ammonia lyase showed increases of 78.37, 114.67 and 125.11% respectively in the resistant variety. Higher expression of glucanase genes was evident from native gel analysis, in which not only the number of isozymes but also the quantity of individual isozymes was significantly increased. CONCLUSION: The resistant variety Dinaar had strong antifungal resistance and can therefore be recommended as suitable for cultivation in the agricultural system of Pakistan.

Cloning and characterization of prophenoloxidase A3 (proPOA3) from Culex pipiens pallens.

The prophenoloxidase subunit A3 (proPOA3) gene was cloned from Culex pipiens pallens, which had an open reading frame of 2061 bp encoding a putative 686 amino acid protein. The deduced amino acid sequence shares 98% with proPOA3 from Culex quinquefasciatus. ProPOA3 is expressed at all developmental stages of C. pipiens pallens. Significant negative correlation was observed between proPOA3 expression and deltamethrin resistance in resistant C. pipiens pallens. Furthermore, proPOA3 expression levels were significantly lower in deltamethrin-resistant mosquitoes than in susceptible mosquitoes collected at four locations in Eastern China. However, we did not find any substantial change in proPOA3 expression in field-collected resistant Anopheles mosquitoes. Moreover, overexpressing proPOA3 in C6/36 cells led to more sensitivity to deltamethrin treatment. In laboratory and field-collected resistant C. pipiens pallens, a valine to isoleucine mutation (769G>A) and two synonymous mutations (1116G>C and 1116G>A) were identified in proPOA3. In addition, the mutation frequency of 769G>A and 1116G>C increased gradually, which corresponded with raised deltamethrin resistance levels. Taken together, our study provides the first evidence that proPOA3 may play a role in the regulation of deltamethrin-resistance in C. pipiens pallens.

Enhancement of biocontrol efficacy of Pichia carribbica to postharvest diseases of strawberries by addition of trehalose to the growth medium.

The effects of trehalose on the antagonistic activity of Pichia caribbica against Rhizopus decay and gray mold decay of strawberries and the possible mechanisms involved were investigated. The proteomic analysis and comparison of P. carribbica in response to trehalose was analyzed based on two-dimensional gel electrophoresis. The antagonistic activity of P. carribbica harvested from the culture media of NYDB amended with trehalose at 0.5% was improved greatly compared with that without trehalose. The PPO (Polyphenoloxidase) and POD (Peroxidase) activity of strawberries treated with P. carribbica cultured in the NYDB media amended with trehalose at 0.5% was higher than that of the strawberries treated with P. carribbica harvested from NYDB. The ß-1, 3-glucanase activity of strawberries treated with P. carribbica cultured in the NYDB media amended with trehalose at 0.5% was also higher than that of the strawberries treated with P. carribbica harvested from NYDB and the control. Several differentially expressed proteins of P. carribbica in response to trehalose were identified in the cellular proteome, most of them were related to basic metabolism.

Enzyme characterisation, isolation and cDNA cloning of polyphenol oxidase in the hearts of palm of three commercially important species.

Heart of palm (palmito) is the edible part of the apical meristem of palms and is considered a gourmet vegetable. Palmitos from the palms Euterpe edulis (Juçara) and Euterpe oleracea (Açaí) oxidise after harvesting, whereas almost no oxidation is observed in palmitos from Bactris gasipaes (Pupunha). Previous investigations showed that oxidation in Juçara and Açaí was mainly attributable to polyphenol oxidase (PPO; EC 1.14.18.1) activity. In this study, we partially purified PPOs from these three palmitos and analysed them for SDS activation, substrate specificity, inhibition by specific inhibitors, thermal stability, optimum pH and temperature conditions, Km and Ki. In addition, the total phenolic content and chlorogenic acid content were determined. Two partial cDNA sequences were isolated and sequenced from Açaí (EoPPO1) and Juçara (EePPO1). Semi-quantitative RT-PCR expression assays showed that Açaí and Juçara PPOs were strongly expressed in palmitos and weakly expressed in leaves. No amplification was observed for Pupunha samples. The lack of oxidation in the palmito Pupunha might be explained by the low PPO expression, low enzyme activity or the phenolic profile, particularly the low content of chlorogenic acid.

A wounding-induced PPO from cowpea (Vigna unguiculata) seedlings.

Polyphenol oxidases (PPO) are induced in cowpea plants by wounding. The highest activity levels were detected 48h after this stimulus in both wounded and neighbor-to-wounded unifoliates of cowpea seedlings; the increase of activity was in the order of 13 to 15-fold, respectively, in comparison to control unifoliates. Multiple molecular forms of active PPO (Mrs 58, 73 and congruent with220kDa) were detected by partially denaturing SDS-PAGE. Wounding-induced cowpea PPO were extracted and purified through (NH4)2SO4 precipitation and ion-exchange chromatography. The effects of substrate specificity, pH, thermal stability and sensitivity to various inhibitors - resorcinol, EDTA, sodium azide and tropolone - of partially purified soluble PPO were investigated. Purified wounding-induced cowpea PPO (wicPPO) showed the highest activities towards 4-methylcatechol (Km=9.86mM, Vmax=24.66 EU [DeltaAmin(-1)]) and catechol (Km=3.44mM, Vmax=6.64 EU [DeltaAmin(-1)]); no activity was observed towards l-tyrosine, under the assay conditions used. The optimum pH for wound-induced cowpea PPO was 6.0 with 4-methylcatechol as substrate. The enzyme was optimally activated by 10 mM SDS and was highly stable even after 5 min at 80 degrees C. The most effective inhibitor was tropolone, whereas addition of 10mM of resorcinol, EDTA and sodium azide were able to reduce PPO activities by 40%, 15% and 100%, respectively.

A prophenoloxidase from the Chinese mitten crab Eriocheir sinensis: gene cloning, expression and activity analysis.

Prophenoloxidase (proPO) is a conserved copper-containing enzyme that plays important roles in immune response of crustaceans and insects. In the present study, the full-length cDNA of a prophenoloxidase (designated EsproPO) was cloned from haemocytes of Chinese mitten crab Eriocheir sinensis by expressed sequence tag (EST) and PCR techniques. The isolated 3549bp full-length cDNA of EsproPO contained a 2040bp open reading frame (ORF) encoding a putative proPO protein of 679 amino acids, a 5'-untranslated region (UTR) of 68bp, and a long 3'-UTR of 1441bp. Two putative copper-binding sites, a proteolytic activation site, and a complement-like motif (GCGWPQHM) were identified in the deduced amino acid sequence of EsproPO. Homology analysis revealed that EsproPO was highly similar to other proPOs from crustaceans with identities from 52% to 68%. The conserved domains and motifs, and higher similarity with other proPOs suggested that EsproPO was a member of the proPO family. The mRNA expression of EsproPO and PO specific activities in the tissues of hepatopancreas, gill, gonad, muscle, heart, eye and haemocytes were measured by quantitative real-time PCR and colorimetric assay, respectively. The mRNA transcripts of EsproPO and PO specific activities could be detected in all the examined tissues with the highest level both in hepatopancreas. Three peaks of EsproPO mRNA expression were recorded at 2h, 12h and 48h in haemocytes of Chinese mitten crab post Vibrio anguillarum challenge, which was consistent with the temporal profile of PO specific activity. The mRNA expression pattern and the activity fluctuation of EsproPO post V. anguillarum stimulation indicated that it was potentially involved in the acute response against invading bacteria in Chinese mitten crab.

Changes in immune gene expression and resistance to bacterial infection in lobster (Homarus gammarus) post-larval stage VI following acute or chronic exposure to immune stimulating compounds.

Real-time PCR was used to measure changes in transcript abundance of genes encoding important immune proteins, namely prophenoloxidase (proPO gene), beta-1,3-glucan binding protein (betaGBP gene) and a 12.2 kDa antimicrobial peptide (amp gene) in post-larval stage VI (PLVI) juveniles of the European lobster, Homarus gammarus. Gene expression was studied in both healthy PLVI and following single or repeat exposure to a range of compounds claimed to induce immune reactivity. A single acute (3-h) exposure to any of the tested stimulants did not produce a significant increase in expression of either the proPO or betaGBP genes, measured 6h after stimulation. However, there were a small sub-group of positive responders, identified mainly from betaGBP expression, within the experimental groups stimulated with either a beta-1,3-glucan or an alginate. There was also no significant increase in the expression of any of the three genes tested 24 h after repeated weekly (3-h) exposures to a either the beta-1,3-glucan or the alginate over the longer (36-day) period. The results do show that amp is expressed at an extremely high level compared to proPO or betaGBP in healthy animals and a significant correlation was found between the expression of proPO and both betaGBP and amp, irrespective of whether or not the larvae were stimulated. None of the immune stimulated compounds improved survival of PLVI challenged with the opportunistic pathogen, Listonella anguillarum, or the lobster pathogen, Aerococcus viridans var. homari. Thus, we found no evidence to support recent claims that immunity and disease resistance can be primed or promoted within a given population of crustaceans or that these animals exhibit functional immune memory to some soluble immune elicitors.

Demonstration of a true phenoloxidase activity and activation of a ProPO cascade in Pacific oyster, Crassostrea gigas (Thunberg) in vitro.

The prophenoloxidase (ProPO) system is the origin of melanin production and is considered to be an innate defence mechanism in invertebrates. In different bivalve species, phenoloxidase (PO) is present in the haemolymph as an inactive form of ProPO. The present study focuses on the Pacific adult oyster, Crassostrea gigas, an economically important bivalve species along French coasts. The results indicate that many factors may inhibit the PO-like activity. These include: phenylthiourea (PTU), sodium diethylthiocarbamate (DETC), beta-mercaptoethanol and tropolone, which repressed the spontaneous PO activity. The activation of PO-like activity in C. gigas acellular fraction by lipopolysaccharide (LPS) involved participation of other factors, including at least one serine protease. PO was present as proPO in the acellular fraction of haemolymph and haemocytes of C. gigas and could be activated by an exogenous protease (trypsin-N-tosyl-l-phenylalanine chloromethyl ketone) when used at 1 gL(-1). Treatment of acellular fractions with other modulators/activators namely LPS (1 gL(-1)), zymosan (0.6 gL(-1)) or laminarin (0.6 gL(-1)) also increased PO-like activity but to a less important way. The study demonstrated the evidence of a true phenoloxidase activity in Pacific oyster, C. gigas (Thunberg). The activation of a proPO system by non-self molecules suggests the role played by PO in vivo in the internal defence mechanisms. Understanding the activation of the ProPO system could enable the evaluation of the health of oyster stocks.

Molecular cloning and characterisation of prophenoloxidase cDNA from haemocytes of the giant freshwater prawn, Macrobrachium rosenbergii, and its transcription in relation with the moult stage.

Expression of prophenoloxidase (proPO) cDNA was determined from haemocytes of the giant freshwater prawn Macrobrachium rosenbergii by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA using oligonucleotide primers based on the proPO sequence of tiger shrimp Penaeus monodon, freshwater crayfish Pacifastacus leniusculus, green tiger shrimp Penaeus semisulcatus, kuruma shrimp Marsupenaeus japonicus, and white shrimp Litopenaeus vannamei. The proPO of M. rosenbergii was constitutively expressed. The 2,547-bp cDNA contained an open reading frame (ORF) of 2,013 bp, a 96-bp 5'-untranslated region, and a 438-bp 3'-untranslated region containing the poly A tail. The molecular mass of the deduced amino acid (aa) sequence (671 aa) was 76.7 kDa with an estimated pI of 7.05. It contained putative copper-binding sites, a complement-like motif (GCGWPRHM), a proteolytic activation site, and a conserved C-terminal region common to all known proPOs. However, no signal peptide sequence was detected in giant freshwater prawn proPO. Comparison of amino acid sequences showed that prawn proPO is similar to the proPO of penaeid, crayfish and lobster. Prawn proPO was only synthesised in haemocytes. The proPO transcript was significantly increased in the A stage and achieved the highest level in the B stage, and then declined sharply in the C stage and reached the lowest level in the D(2)/D(3) stage.