Molecular Cloning, Tissue Expression Of MSTN And MHC ⅡB From Spotted Halibut (Verasper Variegatus)

Spotted halibut(Verasper variegatus)is distributed in China Yellow Sea and East China Sea,south of Hokkaido to kyushu coast. It is popular with the majority of consumers and farmers with delicious meat,rich nutrition and high economic value.Based on the strategy of Homology Cloning and Genome Waking,myostatin(MSTN) partial genomic sequence were amplified from DNA of spotted halibut. The length of MSTN partial genomic sequence is 2992bp,including a 5′flanking region of 873bp and translated region of 1086 bp encoding a polypeptide of 362 amino acids;the myostatin protein contained three domains,a putative signal peptide of 22 amino acids,a conserved proteolytic processing site(RVRR)and 7 cysteine residues in the bioactive carboxy terminal region;the sequence analysis also showed the upstream region contained two TATA boxes,one CAAT box and some transcriptional response factor(AP﹑Sp1﹑MyoD﹑CTF). Besides,homology sequence alignment demonstrated that the MSTN amino sequence shared high homology with other species;Phylogenetic analysis showed that all the teleost clustered into a cluster and other vertebrates clustered into other cluster.In addition,we used rapid amplification of cDNA ends(RACE) approach to get the Major histocompatibility complex (MHCⅡB) cDNA sequence;MHCⅡB has length of 1144bp (Acc. No. GU253881),including a 5′UTR of 7bp,a 3′UTR of 450bp,and an open reading frame(ORF) of 687bp encoding a polypeptide of 228 amino acids,which contained a signal peptide , a peptide-binding region(β1),an immunoglobulin-like region(β2),a transmembrane region and a cytoplasmic region;the homology sequence alignment revealed that the amino of the spotted halibut MHCⅡB shared high homology (30%~79%) with other teleosts and vertebrates,relatively low degree homology was showed in Mus musculus,Homo sapiens,Gallus gallus,Ginglymostoma cirratum;Phylogenetic analysis showed that all the teleost clustered into a cluster and elasmobranchia clustered into other cluster with other vertebrates.We also used Realtime PCR analysis to detect the expression characteristics of MHCⅡB in nine tested tissues(blood,kidney,spleen,heart,skin,muscle,liver,gill and brain). Results showed that MHCⅡB was expressed in nine tested tissues,but the expression levels were different. The expression levels in kidney is the highest,while in muscle is the lowest.