碩士 / 國立臺灣大學 / 動物學研究所 / 97 / Nitric oxide (NO) is an essential molecule involved in neuron transduction, cardiac disease, and immune response while nitric oxide synthase (NOS) is a critical enzyme that catalyzes it. Although numerous researches have been carried out to understand NOS, its role is still unclear in invertebrates including crustaceans.
4,5-Diaminofluorescein (DAF-2DA) staining indicated that shrimp hemocytes could produce NO. However its production can be blocked by NOS specific inhibitor, NG-monomethyl-L-arginine, monoacetate salt (L-NMMA). This reveals that the production of NO in hemocytes is catalyzed by NOS.
We cloned a cDNA of nitric oxide synthase from Penaeus monodon hemocytes (PmNOS) was cloned by nested-reverse transcription-polymerase chain reaction (nested RT-PCR) and 5’- and 3’-rapid amplification of cDNA ends (5’-RACE, 3’-RACE). The full length cDNA of PmNOS includes 249 bp of 5’UTR, 3582 bp of ORF and 166 bp of 3’UTR. The putative peptide is 1193 amino acid residues in length, with an estimated molecular weight of 134.7 kDa and pI 6.7. PmNOS contains oxygenase domain, calmodulin binding motif and reductase domain from N- to C-termini. The oxygenase domain has heme and BH4 binding motifs while the reductase domain contains FMN, FAD and NADPH binding motifs.
Phylogenetic analysis via MEGA4 indicated that PmNOS clusters with known invertebrate NOS, but does not cluster with iNOS, eNOS or nNOS found in vertebrates.
PmNOS mRNA was expressed in many tissues of P. monodon including the thoracic and ventral ganglia, hemocytes, eyestalk, midgut, gill, and subcuticular epithelium as demonstrated using RT-PCR.
The activity of NOS crude extract from different tissue was determined using Griess assay. NOS activities were demonstrated from high to low in that order from different tissues such as thoracic and ventral nerve, subcuticular epithelium, hemocytes and muscle.
RT-PCR revealed that CpG-ODN 2006 did not induce NOS mRNA increase in hemocytes in vitro, neither LPS stimulated NOS mRNA increase in thoracic and ventral nerve, subcuticular epithelium and hemocytes in vivo at any time point.
The recombinant PmNOS was synthesized using baculovirus-insect cell system and its activity confirmed by Griess assay.
PmNOS is noninducible in transcription which is also true with nNOS or eNOS. In addition, the highest NOS activity of thoracic and ventral nerve implies that PmNOS may resemble the nNOS. However, NO production in hemocytes indicates that the function of PmNOS approaches that of iNOS. Since PmNOS has not been categorized in any kind of vertebrate NOS but the transcriptional messages were found expressed in hemocytes, subcuticular epithelium and thoracic and ventral nerve of P. monodon, therefore we suggest that PmNOS may be a prototype of NOS.
| Identifer | oai:union.ndltd.org:TW/097NTU05312001 |
| Date | January 2009 |
| Creators | Chun-Hung Wu, 吳峻弘 |
| Contributors | 宋延齡 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 67 |
Page generated in 0.0171 seconds