| The large yellow croaker (Pseudosciaena crocea, Richardson,1864) is one of the most economically important marine fish species cultured in the coast in the temperate zone, especially in Fujian and Zhejiang provinces. In recent years, with the rapid development of the P. crocea culture industry, serious epidemic caused by viruses, bacteria and parasites are becoming more and more severe and resulting in great economic losses. At present, little information is available on its basal immune response and physiological processes of this fish. In order to obtain important differentially expressed genes involved in physiological and pathological processes, we constructed the cDNA library of head kidney of P. crocea with PG as immunostimulant by the suppression subtractive hybridization (SSH) technique. Two important molecules (BPI,DP1) involved in immune response and physiological process respectively of this P. crocea were studied in detail. Main results of this dissertation included the following three parts:1 Construction of SSH cDNA library of head kidney from large yellow croaker (Pseudosciaena crocea)A subtracted cDN A library was constructed from the head kidney of P. crocea which had been challenged with PG. About 196 randomly-selected clones containing inserts were sequenced. After removing the vector sequence and trimming the poor-quality sequence, we obtained 149 qualified ESTs, which were submitted to GenBank database with accession numbers EB643250-EB643371 and GO271613-GO271637. Tentative annotations were performed by using BLASTX and results were manually validated.26 ESTs were assigned to a defined annotation,22 ESTs with low similarity, whereas 101 ESTs remained unknown. Among the annotated 26 ESTs, five ESTs belong to immune-related genes, three ESTs belong to mitochondrial genes, five ESTs belong to protein kinase, three ESTs associate with signal transduction. In addition, there are two oncogenes, two microsatellites, one male sterility gene, two cytoskeleton genes, two RNA association genes and one ribonucleoprotein gene.2 Cloning, characterization and functional analysis of P. crocea BPI-like molecule (Pc-BPI)A novel homologue, BPI-like molecule (Pc-BPI), similar to lipopolysaccharide-binding protein/bactericidal permeability-increasing (LBP/BPI) was identified. The full length cDNA for Pc-BPI was 1919 nucleotides encoding a protein of 472 amino acids (aa). The deduced molecular weight was 51.28 KDa and the theoretical pI was 5.06. The 5'and 3'untranslated regions (UTR) contain 103 bp and 397 bp, respectively. There is a typical polyadenylation signal (AATAAA) located at 23 bp upstream of the poly (A) tail, and three mRNA instability motifs (ATTTA) were found in 3'-UTR. The BLASTP search showed that the overall amino sequence of large yellow croaker Pc-BPI shared 36%,36%,36%,34%,28% and 25%of residue identity with the channel catfish, common carp, rainbow trout, Atlantic cod, cattle and human BPI/LBP, respectively. SMART result showed two putative functional domains at the position 26-244-aa and 259-461-aa of Pc-BPI that share similarity with BPI/LBP/CETP N-terminal domain (5.02e-31) and BPI/LBP/ CETP C-terminal domain (1.93e-32) found in many mammals. Just like mammals BPI/LBP counterparts, the conserved LPS-binding domain also contained with positive amino acids, indicates that Pc-BPI might bind to lipid A of LPS and plays bactericidal action and endotoxin neutralization activity.The expression of Pc-BPI gene in various tissues was analyzed by RT-PCR during induction with the formalin-inactivated bacterial vaccines. It was found that the Pc-BPI was constitutively expressed in all tissues sampled although at different levels, and its expression was obviously up-regulated in the kidney, intestine, muscle, gills, spleen and head kidney after induction with the bacterial vaccines. These results indicate that the mRNA level of Pc-BPI can be induced.The plasmids containing the fragments corresponding to the N-terminal and C-terminal regions of Pc-BPI were constructed respectively to express each recombinant protein in Eschericia coli, and at the same time, the holo protein of Pc-BPI was expressed in Tn-5B1-4 cell of the cabbage looper moth, Trichoplusia ni by using Bac to Bac system. Recombinant fusion N-terminal region protein was purified by His affinity columns, and purified proteins were used to immunize rabbits to produce antibody. Recombinant fusion N-terminal region protein has shown intensive killing of the bacteria Vibrio harveyi, and also has cytotoxic to Vibrio alginolyticus and Vibrio parahamolyticus which are the pathogens of sicked fish.In addition, the results from immunohistochemistry shown that the Pc-BPI was ubiquitous expressed in spleen, head kidney, kidney and was predominantly located in cytoplasm of parenchymal cells of these organs. There were also observed light positive staining in the nuclei. 3 Cloning, characterization and functional analysis of P. crocea DP1 molecule (Pc-DP1)This paper report here for the first time that a homologue of DPI transcription factor, Pc-DP1, was identified and functionally characterized in the P. crocea (an accession number of DQ 821446). The full-length cDNA of Pc-DP1 is 1427 base pair (bp) long, including 5'and 3'-untranslated regions (UTR) of 116 bp and 70 bp, respectively. The complete open reading frame (ORF) (117-1355) encodes a 412 residue protein with 45.72 kDa molecular weight and the theoretical pI is 6.21. The BLASTP search showed that the overall amino sequence of large yellow croaker Pc-DP1 shared 88%,78%,78%and 78% of residue identity with the zebra fish, frog, human and rat transcription factor DP1 (DRTF1-polypeptide 1), respectively.A comparison of Pc-DP1 with the human DP1 and house mouse DP1 indicated that their DNA binding (113-148) and dimerization domains (149-209) are strikingly conserved. The domain 173-199 (referred as DEF box) which functions to enable a DP protein to interact with an E2F protein, is highly conserved (100% identity) within all known DP proteins, and the consensus residues (RR*YD**NVL*****I*K*K**I*W*G) are strikingly conserved between X. laevis DP1, murine DP1 and human DP1 and also conserved within the other human E2F proteins, E2F1,2,3. These consensus residues may contribute to a leucine zip-type dimerization. All these sequence similarities indicate that Pc-DP1 is a conserved molecule and might can forms a heterodimer with an E2F-like protein and play a vital role in cell-regulating process. In addition, the N-terminal region of Pc-DP1 is rich-in basic residues, whereas the C-terminal has high proportion of acidic amino acids, which consistent with the observations in X. laevis DPI.At the message level, Pc-DP1 mRNA was constitutively expressed in all tissues examined, including spleen, head kidney, kidney, intestine, liver, gills, muscle and heart. And a high level was seen in spleen, intestine and heart, and the lowest level in head kidney.The recombinant Pc-DP1 protein was successfully expressed in E. coli, and purified, as well as the biological activity of the recombinant protein was determined. The antibody against Pc-DP1 was also raised. The subcellular location of Pc-DP1 by immunohistochemistry shown that the Pc-DP1 was ubiquitous expressed in spleen, head kidney, kidney and gills, and Pc-DP1 was predominantly located in cytoplasm of parenchymal cell.EMS A was carried out using DIG-labeled probe containing consensus E2F-site to test the biological activity of the recombinant Pc-DP1 protein. A specific binding activity was observed with recombinant Pc-DP1 protein, demonstrating that the recombinant Pc-DP1 protein has biological activity to bind the consensus E2F-site. This result indicates that the P. crocea transcriptional factor Pc-DP1 has a functional similarity with other DP1 family members. The conservation and constitutively expression pattern of Pc-DP1 strongly suggest the diversity in function of Pc-DP1. |
