Study On ESTs And Cloning, Expression Of Genes Related To Immunity Of Haliotis Discus Hannai

A cDNA library from Haliotis discus Hannai was constructed and expressed sequence tags were sequenced. Three genes related to immunity were cloned using RACE such as NPY receptor, Galectin and AIF-1. 15 microsatellite loci were obtained, and genetic diversity was analysed in 2 cultured populations of Haliotis discus Hannai. The main results are introduced as follows:(1) The cDNA library of the liver and kidney from the Vibrio anguillarum-infected Haliotis discus Hannai was constructed. The content of the library was 5.24×10~5 cfu/mL and the size of the inserted fragments ranged from 0.8 Kb to 2.5 Kb. After random selection of the clones for sequencing, 1282 good qualitive ESTs were obtained. Through the use of Blastx and Blastn, 393 (45%) ESTs shared significant homology with known sequences in protein or nucleotide database of NCBI and 63 genes related to immunology were obtained.(2) A full-length cDNA of the NPY (neuropeptide Y) receptor was cloned with the specific primers based on the sequence of the EST. The full-length cDNA of the NPY receptor from Haliotis discus hannai was 1534bp and encoded 350 amino acid. BLAST analysis revealed that the NPY receptor gene from Haliotis discus hannai shared high identity with the NPY receptor genes from other organisms in amino acid. This NPY receptor have a predicted molecular of 39.56 kDa and theoretical isoelectric point of 9.57, receptively. The NPY receptor from Haliotis discus hannai has many properties of G-protein, for example 7 Transmembrane helices et al. The temporal expression of NPY receptor genes in healthy and Vibrio anguillarum challenged Haliotis discus hannai was measured by semi-quantitative reverse transcription-PCR. The expression of Galectin was up-regulated gradually after stimulation, and reached its maximum level at 12 h timepoint then dropped progressively to the original level.(3) Galectin and AIF-1 cDNA were cloned through the same RACE technique with the specific primers based on the sequence of the EST. The full length of the Galectin cDNA from Haliotis discus hannai was of 2889 bp, including a 5′-untranslated region(UTR) of 84 bp, a 3′-UTR of 1868 bp, and an ORF of 921bp encoding a polypeptide of 307 amino acids with an estimated molecular mass of 34.27 kDa and an estimated isoelectric point of 7.098. The expression of Galectin was up-regulated gradually after stimulation, and reached its maximum level at 12 h timepoint then dropped progressively to the original level.(4) The full length of the AIF-1 cDNA from Haliotis discus hannai was of 1302 bp ,including a 5'-untranslated region(UTR) of 42 bp, a 3′-UTR of 804 bp, and an ORF of 456bp encoding a polypeptide of 151 amino acids with an estimated molecular mass of 17.14 kDa and an estimated isoelectric point of 4.845. The expression of AIF-1 was up-regulated gradually after stimulation, and reached its maximum level at 8 h timepoint then dropped progressively to the original level.(5) Through the use of the Tandem repeats finder,the ESTs database of Haliotis discus hannai was screened. 143 Microsatellite sequences were obtained from 1282 ESTs sequences, about 11.15% in whole database. A total 104 dinucleotide repeats and 19 trinucleotide repeats were found in these 143 microsatellite sequence. In these microsatellite loci, genetic diversity was analysed in 2 cultured population. The average number of allele(A) ranged from 2 to 13.5; the averge heterozygosity (Ho) in 2 population was 0.1635 to 0.9845; averge excepted herterozygosity(He) was 0.317 to 0.8975.