Molecular Cloning, Expressnion And Sequence Analysis Of Myostatin In Four Cultured Marine Flatfishes

In this study the MSTN sequence were cloned from Japanese flounder (Paralichthys olivaceus) by homology cloning approach, RACE PCR and Anchored PCR. The open reading frame is 1134 bp, encoding a protein of 378 amino acids with a signal peptide, 9 conserved cysteine residues and a RXXR proteolytic cleavage domain. Nucleotide sequence analysis of the 5'flanking sequences revealed a'TATA'boxe and 12 E-boxes upstream from the transcription start site. The poly-A signal sequence (AAUAAA) was identified in the 3'UTR. The microsatellite regions were also revealed in the 5'upstream region, intron2 and 3'UTR. RT-PCR showed that the gene had a wide distribution of expression in all 13 tested tissues. It demonstrated that high expression was detected in eyes, brain, skeletal muscle, cartilage, ovary, testes and kidney, low in heart, liver, spleen, gills, blood and intestine. Also the differential expression was observed in different parts of brain. Real-time RT-PCR analysis suggested that the expression of pMSTN appeared to be developmentally regulated.In order to find out proper endogenous control genes for Paralichthys olivaceus embryonic development research, eight commonly used reference genes were choosed to investigate the changes of their RNA transcription levels during the whole embryonic developmental stages. The results showed that all mRNA genes exhibited the significant expression variations and were unsuitable to be used as normalizer throughout the whole embryonic developmental stages. However 18S rRNA displayed the highest stability with no more than 2 fold change during the whole developmental stages. Therefore from our opinion, 18S rRNA could be used as a suitable normalizer thoughout embryonic developmental stage.Analyzing the 60 MSTN sequences from 20 different Japanese flounder revealed 19 SNPs sites existing within the CDS. Eight of them were neutral without causing changes in amino acid sequences, and the rest of them caused changes in amino acid sequences. Also we analyzed the polymorphism of two microsatellite sites within the intron2 and 3'-UTR in two cultured Paralichthys olivaceus populations (pairmating and groupmating) among which some fish growed slower than others, however, no dominant difference were found between the fast-growing one and the slow-growing one. This information indicated that these two microsatellites sites maybe have no direct relationship with the growth of Paralichthys olivaceus.No significant effect of dead V.anguillarum injection on the expression of MSTN in muscle, liver and kidney were observed (p>0.1). Despite of statistical non-significance, the elevations of the expression of MSTN in liver and kidney with dead V.anguillarum injection were occurred when compared with mock injection. We suggested that the little increase of MSTN expression observed in injection group likely was induced by the injury of liver or kidney caused by the stimulation of dead V.anguillarum, just as occurred in cardiomyocytes after the infarction, in which myostatin expression was upregulated.Three MSTN sequences were cloned from stone halibut(Kareius bicoloratus), turbot(Scophthalmus maximus)and tongue sole(Cynoglossus semilaevis) by homology cloning approach and RACE PCR. The stone halibut MSTN is 2457 bp long and consisted of 50 bp 5'UTR, 1134 bp open reading frame, 1273 bp 3'UTR, 348bp intron1 and 703bp intron2. Turbot MSTN is 2559 bp long and consisted of 48 bp 5'UTR, 1131 bp open reading frame, 1380 bp 3'UTR, 254bp intron1 and 845bp intron2. Tongue sole MSTN is 1584 bp long and consisted of 49 bp 5'UTR, 1134 bp open reading frame, 401 bp 3'UTR, 740bp intron1 and 403bp intron2. The poly-A signal sequence (AAUAAA) was identified in the 3'UTR of each MSTN genes. All exon-intron boundary sequences conform to the GT-AG rule, and the microsatellite regions were also identified in these introns and 3'UTRs.The propeptide and the mature peptide were cloned into pGex-4T-3-His espression vector to produce two different recombinant plasmids with different Japanese flounder MSTN domains. The recombinant plasmids were later introduced into E.coli.BL21 (DE3). 12% SDS-PAGE analysis showed a expression protein band of about 55kDa corresponding to the recombinant propeptide, and a bnad of about 38kDa corresponding to the recombinant mature peptide. The two recombinant proteins were pufified using His-bind pre-packed columns, and the soluble recombinant propeptide was obtained after urea gradient dialysis.