碩士 / 東吳大學 / 微生物學系 / 96 / In previous study, using a pair of degenerate primers based on two highly conserved domains of Toll genes of both Drosophila melanogaster and Caenorhabditis elegans, we attempted to clone the cDNA of Toll gene from hemocytes of Macrobrachium rosenbergii. However, an unknown functional and CpG DNA-specific induced cDNA fragment with 1120 nucleotides was cloned (Chiang, 2006). In this study, we cloned the complete cDNA of the CpG DNA-specific induced gene from hemocytes, and determined it’s evolutional phylogenetics, gene expression and the signaling pathway triggered by CpG DNA.
In the first step of this study, prawn treated with different immunostimulants (included ODN2006, LPS and β-glucan) at 6 hr to confirm this cDNA fragment is a CpG DNA-specific induced cDNA of an unique gene by RT-PCR analysis. Subsequently, we design specific primer to perform 3’-RACE and 5’-RACE cloning of the CpG DNA-specific induced cDNA. The length of the transcript obtained is 1707 nucleotides with a putative protein of 408 amino acids. There is no reported sequence in NCBI database is similar to the unique gene. However, analysis of this protein sequence by TMHMM Server v. 2.0 analysis system in CBS Prediction Servers web indicates that there is a transmembrane domain in this protein. We presume this gene product maybe a membrane receptor. The results indicate that we cloned an unknown CpG DNA-specific induced cDNA of an unique gene from hemocytes of Macrobrachium rosenbergii.
To understand the function of this unique gene, the tissue-specific expression of the unique gene in various tissue samples, which were extracted at 1, 2, 3, 6 and 9 hrs after ODN2006 treatment, was determined by RT-PCR. The results showed that unique gene is only expressed in brain tissue under usual condition. When ODN 2006 is employed, this unique gene is only expressed in brain, lymphoid organ and hemocyte. The results suggest that the unique gene might play a relevant role in the nervous system and immune defence.
To further understand the regulation of the unique gene in either NF-κB or ERK signal pathway; the four different concentrations of NF-κB and ERK specific inhibitor are employed. Using low-does (5 μM) of PDTC, the specific inhibitor of NF-κB, could reduce the expression of unique gene induced by ODN2006. But treated with the high-does (30 μM) ERK specific inhibitor, PD98059, the expression of unique gene induced by ODN2006 would be reduced apparently. The results suggest that CpG DNA-specific induced cDNA of an unique gene major regulated by NF-κB signal pathway.
Comprehensive above mentioned result, we obtained an unknown CpG DNA- specific induced cDNA of an unique gene and this unique gene is regulated by both NF-κB and ERK signal pathway.
| Identifer | oai:union.ndltd.org:TW/096SCU05381007 |
| Date | January 2008 |
| Creators | Shin-yi Chiang, 姜馨怡 |
| Contributors | Hung-Hung Sung, 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 | 126 |
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