Cloning, Sequencing In Yak CAPN1 Gene And CAST Gene

Calpains are a family of calcium-dependant cysteine-proteases,it widely distributed in the majority of animal cells.Whichμ-calpain protein is a ubiquitous calpain that requires micromolar levels of calcium for proteolytic activity in vitro.its large subunit ecoded by CAPN1 gene. Calpastatin (CAST)is its hight efficiency and specific inhibitor protein. Research shows that theμ-calpain degrades myofibrillar proteins under postmortem conditions and appears to be the primary enzyme in the postmortem tenderization process of frozen meat in storage.Its large subunit of the gene encoding CAPN1 may influence muscle tenderness as the quantitative trait loci (QTL) of candidate genes. Its inhibitors,Calpastatin,that can inhibit its activity and reduce protein hydrolysis, and its ratio of the activity associated with a high degree of tenderness.Our study according to the CAPN1 gene sequence of Cattle in GenBank,the specific primers Were designed.Using the Tianzhu White yak cDNA as template,PCR amplifying,cloning and Sequencing for every fragment.Using the BioEdit to splice sequences and the a 2267bp yak cDNA of CAPN1 gene was obtained.Including a complete open reading frame(ORF) and partical sequence of 3′end and 5'end UTR.Analysis of the gene revealed that the yak CAPN1 gene is encoded by 2151bp and the 716 AA potein predicted from this sequence. The nucleotide sequence and amino acid sequence of yak calpain were compared with those o f cattle, pig, human,and mouse calpain. Identities for nucleotide and amino acid sequences were 99.3 and 99.4% for cattle,93.9 and 96.1% for pig, 90.0and 94.6% for human, and 85.5and 89.0% for mouse calpain. Search using NCBI BLAST of yak four domains respectively found the four domains in the above 4 species have shown good conservative, the most conservative in domain IV (> 96%). There are 14 nucleotide mutations between yak and cattle and three of that were predicted to alter the protein sequence,both of which occurred in the domain III. The CAST gene is encoded by 2361bp and the 786 AA potein predicted from this sequence. .Homogeneous analysis demonstrated that nucleotide and amino acids sequences of the yak CASTgene between different species mammal are comparatively conservative. The hydrophilic nature of the amino acids that compose calpastatin seems to be a highly conserved characteristic in different species.Construction of molecular phylogenetic tree to the two genes respectively that shows : The cluster results coincided with the traditional taxonomy .