| Abalone Haliotis discus hannai and scallop Chlamys farreri are important natural resources in Huanghai and Bohai Sea area of China, but also the importance of culture in northern China. Since the 1990s, the frequent occurrence of diseases, causing economic losses of 10 billion yuan, a direct threat to the survival of existing industries. In order to promote China's shellfish aquaculture and the healthy development, to pursue durative of China's blue agriculture, the immune resistance mechanisms of aquaculture animals become the most prominent and requiring urgent solution.The antibacterial peptides are the gene encoding peptide antibacterial molecules, which are widespread in the organism. They are key congenital immune factor for vertebrates, invertebrates and plants. For a long time, the antimicrobial peptide research was focused on the higher animals and some insects, marine antimicrobial peptide is a hot topic in recent 10 years. To study Haliotis discus hannai and Chlamys farreri as a research objective, This paper carried out antimicrobial peptides research. The results made the following findings:(1) By using solid-phase extraction and HPLC technology, combined with sensitive anti-microbial enzymic activity test, one kind of antimicrobial peptides was purified from Chlamys farreri blood. It has a molecular weight of 2000 Da, some amino acid sequence was determined to GQPGHTGNAH…….(2) Via analysis, 322 nucleotides from the defensin-related EST were identified in the liver and kidney cDNA library of Haliotis discus hannai Ino. Sequence analysis showed that the tailing signal AATAAA located at 3 'poly (A), 5' with ATG initiation codon, which has won the full-length cDNA sequences. In the ORF (open reading frame) of cDNA sequence, full -length 201 nucleotides encoding 66 amino acid residues of the prepropeptide. Analyzing the signal peptide and enzymatic digestion of the coding protein, found that its precursor consist of signal peptide (18 AA), the leader peptide (6 AA) and the mature peptide (42 AA). Submitted mature peptide amino acid sequence analysis proved the amino acid sequence of defensin has high similarity to other insects defensin, and similarity of 70% to the beetles Anomala cuprea.(3) The bacterial stimulation treatment showed that the gene expression is inducible. Under normal circumstances (control group) the defensin gene was not expressed. Gram-positive and Gram-negative bacteria infection can cause the gene expression, but V.anguillarum induced high expression than Staphylococcus aureus.Under artificial infection in the stimulation of different time stages, the hd-def gene expression exist great differences. In the early stage of infection (2 hr) expression is very weak, in process of 4 hr the expression increased significantly, late in the infection (12 hr) hd-def expression is up to the largest volume, but in 0,6,8 and 24 hr the hd-def gene transcriptional expression was not detected.(4) The 4032 bp of the full-length genome sequence was obtained using genome walking. The analysis results showed that the gene consist of three introns and four exons coding, which three intron size were 497, 2357 and 528 bp, two of them were in the presence of the coded signal peptide sequence. This is a new structure model for defense gene. It was not reported in other insects defense.5) The recombinant gene expression vector pPIC9k-hddef of the hd-def gene was successfully constructed by using yeast expression system. In vitro, the expressed 4.3 kd protein of the recombinant yeast has anti-V. anguillarum and anti-E.coli activity. |
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