| Agamaki Clam (S. constricta) is the most important interdial economic mollusc in China, among one of four major aquacultural bivalves along with Crassostrea gigas, Ruditapes philippinarum and Tegillarca granosa. In recent years, the mortality rate is increasing because of various diseases. So increasing disease resistance is considered to be an important solution in reducing diseases of S. constricta. In this thesis, a normalized cDNA library of hepatopancreas from S. constricta was constructed and the immune-relevant genes were identified from the library. Then, their full length cDNA and genomic DNA were obtained using RACE and LA-PCR, and these genes were studied in different tissues and different time after bacteria induced by qRT-PCR analyses. These results were performed in an effort to obtain immune candidate genes from S. constricta and to lay the foundation for further drug eploitation and selecting breeding to prevent disease.1. A normalized hepatopancreas cDNA library from hepatopancreas in S. constricta was constructed by duplex-specific nuclease (DSN) treatment. A total of 5296 ESTs were sequenced, from which 540 contigs and 3473 singletons were identified. BLAST homology analysis indicated only 20.7% of these ESTs were homologues of known genes while the remaining 79.3% appeared to be novel sequences. Based on sequence similarities, 43 putative immune genes were identified that encode proteases and protease inhibitors, adhesive proteins, stress proteins, lysosomal enzymes and signal transduction regulators.2. Two EST sequences with high homology withβ-actin gene of other species were found from the cDNA library, namedβ-ACTIN 1 andβ-ACTIN 2, respectively. The full length cDNA ofβ-ACTIN 1 andβ-ACTIN 2 was 1552 bp and 1454 bp, respectively, which consisted of one 1131 bp open reading frame (ORF). Meanwhile the full DNA of 18S rRNA was obtained by homology-base cloning. The qRT-PCR analyses indicated 18S rRNA gene could be as an internal control since the expression level of 18S rRNA gene was much more stable in different tissues thanβ-ACTIN 1 andβ-ACTIN 2. As 18S rRNA gene being an internal control, the expression level of twoβ-actin gene had significant change in different tissues and different time after the Vibrio anguillarum chanllenge in S. constricta.3. Two EST sequences with high homology with C-type lectin were found from cDNA library. The full length cDNA of these two genes was 644 bp and 1659 bp, named ScCTL-1 and ScCTL-2, respectively. ScCTL-1 gene is a standard long C-type lectin and ScCTL-2 gene is a compact C-type lectin, with a carbohydrate recognition domain(CRD), respectively. The qRT-PCR analyses showed that the expression levels were different about these two genes in all of the test tissues. After S. constricta were challenged with V. anguillarum and V. parahaemolyticus, the ScCTL-1 and ScCTL-2 mRNA expression levels in all of the test tissues had the differrnt expression change in the same time distribution. It suggested that these two C-type lectins from S. constricta might have different function.4. One EST sequence with high homology with galectins was found from cDNA library. The full length cDNA of this gene was 1278 bp with consensus H-NRP or WG-EE motifs thought to play an essential role in sugar-binding, named Sc-Galectin. The deduced protein contained two tandem CRDs like other galectins, indicating that this gene is a member of the tandem-repeat type galectins. The qRT-PCR analyses showed that the expression level of Sc-Galectin mRNA was significantly higher in hepatopancreas than other tissues. V. anguillarum group compared to control group with PBS-challenge at 8 h and 48 h post-challenge, the Sc-Galectin mRNA expression levels in all of the test tissues were significantly up-regulated. Meanwhile, V. parahaemolyticus group compared to control group with PBS-challenge at 72 h and V. anguillarum group compared to control group with PBS-challenge at 20 h, the Sc-Galectin mRNA expression levels in all of the test tissues were significantly suppression, respectively.5. One EST sequence with high homology with C1q was found from cDNA library. The full length cDNA of this gene was 712 bp, named ScC1q. The translated protein contained a signal peptide of 18 amino acids and a globular C1q domain of 136 amino acids. The qRT-PCR analyses showed that the expression level of ScC1q mRNA was significantly higher in hepatopancreas than other tissues. After S. constricta were challenged with V. anguillarum and V. parahaemolyticus, the ScC1q mRNA expression levels in all of the test tissues were significantly up-regulated or down-regulated, and the expression change trends were more significant in water pipe, gill, abdominal foot, mantle and gonad than hepatopancreas.6. One EST sequence with high homology with ferritins was found from cDNA library. The full length cDNA of this gene was 1106 bp, named ScFERs. The deduced ferritin contained a highly conserved motif for the ferroxidase center, which consisted of seven residues of a typical vertebrate heavy-chain ferritin, but lacking of a iron-responsive element (IRE) with a typical stem-loop structure in the 5’UTR position. The qRT-PCR analyses showed that the expression level of ScFERs mRNA was significantly higher in hepatopancreas than other tissues. After S. constricta were challenged with V. anguillarum and V. parahaemolyticus, the ScFERs mRNA expression levels in all of the test tissues were significantly up-regulated or down-regulated, and the expression change trends were more significant in mantle, abdominal foot and gonad than water pipe, gill and hepatopancreas.7. One EST sequence with high homology with heat shock protein 70 gene was found from cDNA library. The full length cDNA of this gene was 2335 bp, which is a member of cytoplasmic HSC70 (constitutive genes) subfamily in the HSP70 family, and was designated as ScHsc70. The qRT-PCR analyses showed that ScHsc70 mRNA expressed constitutively in all of the test tissues, and the lowest expression level of ScHsc70 mRNA was detected in mantle, the highest expression level was in water pipe. After S. constricta were challenged with V. parahaemolyticus and V. anguillarum, the ScHsc70 mRNA expression levels in hepatopancreas were up-regulated, and the expression was maximum after 20 h and 30 h, respectively. The results suggested that ScHsc70 might play an important role in mediating the immune responses of S. constricta to bacterial infection.8. One EST sequence with high homology with small heat shock protein gene was found from cDNA library. The full length cDNA of this gene was 1220 bp, which has a hallmark of the sHSP subfamily ofα-crystallin domain, and is designated as Sc-sHSP. The qRT-PCR analyses showed that Sc-sHSP mRNA expressed constitutively in all of the test tissues, and the highest expression level was in hepatopancreas. After S. constricta were challenged with V. parahaemolyticus and V. anguillarum, the Sc-sHSP mRNA expression levels in hepatopancreas were up-regulated, and the expression was maximum after 20 h. The results suggested that Sc-sHSP might play an important role in the immune response of S. constricta to bacterial infection. |
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