Molecular Cloning, Expression Of Beta Actin And G-type Lysozyme Genes From Mizuhopecten Yessoensis

Scallop aquaculture is a big industry and contributes enormously to the economic development of coastal provinces in China. In these years, large-scale mortality of cultured scallop has caused catastrophic losses to scallop aquaculture, which resulted in the production decreasing drastically. The durative outbreak of diseases has stimulated intensive efforts for the development of better health management strategies and characterization of original immune efforts for disease control. The identification and characterization of genes involved in scallop immune responses are now considered to be essential for the elucidation of immune defense mechanisms and disease control.in this research, the cDNA library from mantle and kidney of mizuhopecten yessoensis was constructed using Creator SMART cDNA construction kit and large numbers of colonies were randomly picked and sequenced from the 5'. One EST with high homology withβ-actin gene of other species was found and then the complete express sequence ofβ-actin from mizuhopecten yessoensis was obtained by PCR. The cDNA of this gene was 1536bp with 1131 bp of ORF, which encodes 177 amino acids. The intron of this gene was 1498bp which cited between the 41st amino and the 42nd amino. The gene in this study is used for other genes'quantitative expression of mizuhopecten yessoensis as an intemal control.The other EST is also found with high homology with G-type lysozyme genes of other species. and then the complete express sequence of G-type lysozyme genes from mizuhopecten yessoensis was obtained by PCR. The cDNA of this gene was 731bp with 606 bp of ORF, which encodes 202 amino acids. The gene has five introns which lengths are 7087 bp,5178 bp,3526 bp,710 bp and 1400 bp which cited the extron into 6 fragments which lengths are 55 bp,60 bp,90 bp,113 bp,148 bp and 140 bp. The expression of G-type lysozyme in various tissues was measured by using real time PCR analysis. The temporal expression of G-type lysozyme in haemolymph after Vibrio anguilarum challenge was recorded by quantitative real time PCR. The relative expression level of G-type lysozyme in haemolymph was up-regulated evenly in the first 9 h, followed by a drastic increase. Meanwhile, we found several SNPs in the G-type lysozyme mRNA of mizuhopecten yessoensis.Through the method of High-throughput SNP genotyping by single-tube PCR with Tm-shift primers, it tune out that there is one Sense mutation in it. Which established the foundation for the study of genetic polymorphism and gene expression.