| The grouper is a popular food fish cultured in Southeast Asia and a potentially important aquaculture species. Among the more than 150 species of grouper worldwide, yellow grouper Epinephelus awoara is one of the major species of high economic value farmed in China. With the rapid development of the E. awoara culture industry, infectious diseases caused by bacteria, viruses and parasites are becoming more and more severe, which leads to great economic losses. At present, little is known about the genetics and immunology of this fish. It is necessary to establish effective measures for disease control and genetic improvement.Suppression subtractive hybridization (SSH) is a powerful approach to identify differentially expressed genes that are involved in physiological and pathological processes. It dramatically increases the probability of obtaining low-abundance differentially expressed cDNAs and simplifies the analyses of the subtracted cDNA libraries. At present, there is still little information about the molecular response of E. awoara to LPS. In this study, we use SSH to identify differentially expressed genes in LPS-stimulated E.awoara spleen and use RACE-PCR to obtain full length cDNAs and expression of some immune-related genes in an attempt to understand the molecular processes involved in bacteria disease primary prevention. Main results of this dissertation included the following four parts:1. Construction of a suppression subtractive hybridization (SSH) libraryA subtracted cDNA library was constructed from the spleen of E. awoara which had been challenged with LPS. The subtraction efficiency was evaluated by the constitutively expressed gene a-tubulin. The amount of a-tubulin was significantly decreased after subtraction. Obvious bands were seen after 23 cycles in unsubtracted cDNA but only after 33 cycles in subtracted cDNA, indicating a high subtraction efficiency. From cloned PCR products,209 randomly-selected clones containing inserts were sequenced. After removing the vector sequence and trimming the poor-quality sequences, we obtained 153 qualified ESTs, and assembled them into 36 contigs and 56 singlets with an average length of approximately 350 bp. They were submitted to GenBank database with accession numbers EB410743-EB410834. Tentative annotations were performed by using BLASTX and results were manually validated.34 sequences were assigned to a defined annotation, whereas three sequences showed similarity to unclassified genes and 55 sequences remained unknown. In order to further validate the differential expression of the genes in the subtracted cDNA library, we also performed RT-PCR analysis of eight genes in the LPS-treated E. awoara and control spleens. All genes except for the unknown protein showed some increase in the expression in the LPS-treated E. awoara. The expression patterns obtained by RT-PCR reflected the results obtained by SSH, demonstrating a low false positive rate associated with SSH in this experiment.2. Cloning and expression of E. awoara defender against cell death 1Among all the identified ESTs, three immune-related sequences were studied further. RACE-PCR was used to obtained full-length cDNAs of three interesting genes. The nucleotide sequence of full-length cDNA of E. awoara defender against cell death 1 (DAD1) was 721 bp and contained an open reading frame of 342 nucleotides, encoding a predicted protein of 113 amino acids. The deduced molecular weight was 12.5 KDa and the theoretical pâ… was 6.69. E. awoara DAD1 was 86% identical to Danio rerio,84% identical to Homo sapiens, Mus musculus and Sus scrofa. E. awoara DAD1 protein had a signal peptide with 39 amino acids. RT-PCR analysis showed an increase in the expression of DAD1 in the spleens of LPS-treated E. awoara, compared with normal E. awoara, which indicates that LPS might be a stimulator of PCD. A phylogenetic tree was constructed using protein sequences of DAD1, which revealed a closer relationship between yellow grouper and teleosts DAD1 proteins than between yellow grouper and mammalian DAD1 proteins.3. Cloning and expression of E. awoara allograft inflammatory factor-1The yellow grouper allograft inflammatory factor-1 (AIF-1) was originally identified from the SSH library of LPS stimulated yellow grouper spleen. We amplify the full-length transcript using 3'-RACE-PCR and 5'-RACE-PCR. The nucleotide sequence of full-length cDNA of E.awoara AIF-1 was 1102bp. The open reading frame is 444 nucleotides, encoding a predicted protein of 147 amino acids. The deduced molecular weight was 16.6 KDa and the theoretical pâ… was 5.57. E. awoara AIF-1 was 84% identical to Pagrus major,82% identical to Takifugu rubripes,66% identical to Homo sapiens, and 64% identical to Mus musculus. RT-PCR analysis showed an increase in the expression of AIF-1 in the spleen and anterior kiney of LPS-treated E. awoara, which indicates that AIF-1 might be involved in inflammation.The gene fragment encoding AIF-1 protein was cloned into the pET28a expression vector. After induction with IPTG and resolved in a SDS-PAGE, the expressed AIF-1 with an approximate molecular weight of 16.6 kDa was readily observed. Recombinant fusion proteins were purified by His-tag affinity columns, and purified proteins were used to immunize rabbits to produce antibody. Western blot analysis was conducted using polyclonal antiserum against AIF-1. A band with the molecular mass of about 16.6 kDa was detected, which correlated well with the size of deduced AIF-1 amino acids sequence.4. Cloning and expression of E. awoara regulators of G protein signalling 16The yellow grouper regulators of G protein signaling (RGS16) was originally identified from the SSH library of LPS stimulated yellow grouper spleen. We amplify the full-length transcript using 3'-RACE-PCR and 5'-RACE-PCR. The nucleotide sequence of yellow grouper RGS16 full-length cDNA was 700 bp and contained an open reading frame of 537 bp, encoding a putative protein of 178 amino acids. The deduced molecular weight was 20.5 KDa and theoretical pâ… was 8.65. Yellow grouper RGS 16 had a single conserved RGS domain with the other members of RGS family. Yellow grouper RGS 16 was found to be 61% identical to zebrafish,49% identical to house mouse,48% identical to human and 47% identical to cattle RGS 16, respectively.The tissue distribution and expression level of mRNA coding for RGS 16 in LPS-treated yellow grouper and normal yellow grouper were analyzed by RT-PCR. RGS 16 expression was detected in normal yellow grouper spleen. After LPS challenge, a substantial amount of RGS 16 was constitutively expressed in the spleen, heart, anterior kidney, kidney and liver. Moreover, the expressions of RGS 16 gene in spleen and liver were significantly higher than other organs 24 h post LPS treatment, which indicates that the RGS 16 protein may be involved in immune reaction. The complete RGS 16 protein was expressed in E.coli. Recombinant fusion proteins were purified by His-tag affinity columns, and purified proteins were used to immunize rabbits to produce antibody. The pET28-RGS16 was also expressed in Bac-to-Bac expression system. Western blotting analysis with anti-RGS16 polyclonal antibodies as the first antibodies confirmed that the bands of 20.5 kD were expressed in E.coil and Tn-5B1-4, respectively.In conclusion, in the present study we were able to demonstrate the application of SSH, RT-PCR and RACE-PCR to isolate and identify differentially expressed genes in the spleens of LPS-treated E.awoara. We finally obtained 92 unique genes, and full length cDNAs defender against cell death 1, allograft inflammatory factor-1 and regulators of G protein signaling 16. The prokaryotic expression vectors of AIF-1 and RGS 16, and eukaryotic expression vector of RGS 16 using Bac-to-Bac system were successfully constructed, and their proteins and polyclonal antibodies were obtained. Further investigations will be required to determine the special functions of these proteins and processes involved in the defense mechanism of the marine fish, E. awoara.
|
PDF Full Text Request