碩士 / 東吳大學 / 微生物學系 / 92 / In crustacean, the prophenoloxidase (proPO) activating system plays an important role in the nonspecific defense system. Our previous studies have proven that, in freshwater prawn Macrobranchium rosenbergii, the PO activity is related to the defense functions and can be enhanced by immunostimulants. The activity is easily changed by many intrinsic and extrinsic factors; therefore, it is not suitable to use the PO activity as a criterion for the activation of proPO system. The purposes of this study was to clone the proPO cDNA from prawn hemocytes and to further establish a surveillance system based on the gene expression to determine the relationship between the expression of the proPO system and defense system of prawn.
For cloning proPO cDNA, several degenerate primers were designed based on conserved regions in amino acid sequences of homologous proPOs. By combining reverse transcriptase-polymerase chain reaction and 3’/5’-RACE (rapid amplification of cDNA ends), the full proPO cDNA with 2141 bp was obtained. The analysis of nucleotide sequences indicated that the complement cDNA contained an open reading frame with 1788 bp and could encode a putative polypeptide with 595 amino acids. Further analyzing the amino acid sequence showed that the polypeptide contained 6 histidine residues and a thiol ester-like motif (GCGWPRHM). By comparing the proPO cDNA nucleotide and amino acid sequence of M. rosenbergii with those of Pacifastacus leniusculus, Penaeus monodon, Marsupenaeus japonicus, Penaeus semisulcatus and Homarus gammarus, the results showed that the similarity of nucleotide sequence was 61.2 %, 61.9 %, 61.6 %, 62.1%, 61.7 % and 61.0 %, respectively; and that the similarity of amino acid sequence was 65.1 %, 68.6 %, 67.6 %, 67.2 %, 68.1 % and 68.5 %, respectively.
To determine the expression of proPO gene, the partial proPO cDNA used as a target gene was specifically amplified with a pair of specific primers designed from the partial proPO cDNA by the semi-quantitative RT-PCR analysis. In the experiment, the actin gene was used as an internal control. The preliminary test showed that the expression of proPO mRNA of hemocytes from PBS-injected prawns was not different from that of un-injected prawn, but the PO activity of PBS-injected group was increased. The results indicate that the expression of proPO gene is not affected by injection. Consequently, the proPO gene expression of prawns injected with 5 mg/100ml ODN2006 showed that the highest level of proPO mRNA was detected at 1 h after injection; as well as, the highest activity of PO was detected at 6 h after injection. These results suggest that, not only for stability but also for timing of analysis, the level of proPO mRNA is a better indicator than PO activity used as a criterion to evaluate the activation of proPO system.
| Identifer | oai:union.ndltd.org:TW/092SCU00381021 |
| Date | January 2004 |
| Creators | 黃彥棟 |
| Contributors | 宋宏紅 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 80 |
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