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.
Liu
Chun-Hung
CH
Department of Aquaculture, National Pingtung University of Science and Technology, 1 Hseuh-Fu Road, Nei-Pu Hsiang, Pingtung County 91201, Taiwan, ROC.
Tseng
Deng-Yu
DY
Lai
Ching-Yi
CY
Cheng
Winton
W
Kuo
Ching-Ming
CM
D000367:Age Factors; D000595:Amino Acid Sequence; D000818:Animals; D004156:Catechol Oxidase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000737:chemistry; D004792:Enzyme Precursors / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D015870:Gene Expression / Q000502:physiology*; D006433:Hemocyanins / Q000737:chemistry / Q000235:genetics; D006434:Hemocytes / Q000201:enzymology* / Q000502:physiology; D043143:Hepatopancreas / Q000502:physiology; D008969:Molecular Sequence Data; D018978:Molting / Q000502:physiology*; D009132:Muscles / Q000502:physiology; D033541:Palaemonidae / Q000201:enzymology / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D010802:Phylogeny; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D017421:Sequence Analysis / Q000662:veterinary
D017931:DNA Primers; D018076:DNA, Complementary; D004792:Enzyme Precursors; D012313:RNA; D006433:Hemocyanins; C038451:pro-phenoloxidase; D004156:Catechol Oxidase