A goose-type lysozyme gene in Japanese scallop (Mizuhopecten yessoensis): cDNA cloning, mRNA expression and promoter sequence analysis
Lysozyme is an important component of the immune response against bacteria that is characterized by its ability to break down bacterial cell-walls. We constructed a high-quality cDNA library from mantle tissue of adult Japanese scallop (Mizuhopecten yessoensis). The EST which is high homology with g-type lysozyme genes of other species was found in the cDNA library. In the present study, the complete express sequence of g-type lysozyme genes from Japanese scallop (designated as MyLysoG) was directly obtained by PCR. The complete sequence of MyLysoG cDNA consisted of a 5' untranslated region (UTR) of 25 bp, an open reading frame (ORF) of 606 bp, and a 3' UTR of 100 bp with one polyadenylation signal (AATAAA). The deduced amino acids of MyLysoG were 201 amino acids with a putative signal peptide of 18 amino acid residues. It shared the sequence similarity and the common structure features with the g-type lysozyme from other species. Quantitative reverse trancriptase real-time PCR (qRT-PCR) assay demonstrated that mRNA transcripts of g-type lysozyme could be detected in various tissues of unchallenged scallop, and the highest expression of MyLysoG was detected in hepatopancreas tissue. The temporal expression of MyLysoG in hemolymph after Vibrio anguillarum challenge was up-regulated and reached the maximum level at 3h post stimulation, and then dropped back to the original level even lower than the control group. Furthermore, a 978 bp of 5'-flanking sequence of MyLysoG was identified by genome walking, and several potential transcription factor binding sites (TFBS) were detected in the putative promoter region. One part of the MyLysoG promoter region contains nine sites of SNPs and three sites of insert-deletion (indel) polymorphisms, and these mutations were found organize into two haplotypes. The two haplotypes were associated with different TFBS. The haplotypes could be selected to analyze the transcriptional-level control of scallop g-type lysozyme gene and the scallop immune system.
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