CDNA Cloning, Tissue Expression And Bioinformatic Analysis Of The Chicken Calpastatin Genes

The calpain system which mainly comprised molecules:two Ca²⁺-dependent proteases,mu-calpain and m-calpain,and a third polypeptide,calpastatin,is ubiquitous in cells,involved in the course of growth and metabolizability of organism,especially in the renovating of the myofibril and postmortem tenderization of skeletal muscle.This system has been implicated as a principal regulator of myofibrillar protein degradation in both ante-mortem and postmortem muscle.Calpastatin is a specific,endogenous inhibitor of the calpains activated by Ca²⁺ inhibiting both forms of calpains(low calcium-requiring form called u-calpain and a high calcium-requiring form called m-calpain),and inhibits the decomposability of protein in muscle and reduces the growth speed of muscle cell.After slaughter,it can inhibit the activity of calpain and depressed the hydrolyzation of protein, therefore,Calpastatin considered to be one of the major modulators of the calpains.The CAST gene were considered as the candidate genes for growth traits and meat color traits.The current research adopted Sichuan Mountainous Land Black-Bone chicken as material.We successfully obtained the coding sequence of CAST gene,which was 2361bp. Based on the cDNA sequence,we deduced one amino acid sequences.It lengths was 761aa. Between products sequences and Gallus(XM₄24713 ),the homology rates of nucleotide sequences of coding region of CAST gene were 99.9%respectively.There was a bases transition as T→C on site of 308 caused an amino transition as Leu→Pro on site of 112. There was a bases transition as T→C on site of 486.Bioinformatic primary analysis suggested that chicken CAST is a plasma membrane protein which has two transmembrane helice and several hydrophobicity domains.The relative molecular weight of chicken CAST is 70.48 KDa,and isoelectric point is 5.49.The amino sequence may have phosphorylation sites,the calpastatin polypeptide is nearly completely in a random coil conformation and it has the similar Crystal structure of the Calpain_inhib.The present study took Sichuan Mountainous Land Black-Bone chicken,Tibetan chicken and line S01 of Daheng chicken as materials.We adopted the real-time PCR (SYBR green I) method to test the expression quantity of CAST gene in the breeds mentioned above at the first day and the 84th day after birth.And we also tested the expression quantity of CAST gene in different growth points in Sichuan Mountainous Land Black-Bone chicken.The results indicated that CAST gene express in all tissues at all growth points,and there was advantageous tissue for gene expression at each growth point and there was also advantageous growth point for gene expression in a certain tissue.And the rule of relative quantities of CAST gene was that all the growth points in breast muscle were highest compared with these in other tissues.CAST mRNA expression in muscle was little low after hatch,but it expression was abundant after birth.CAST mRNA tissues expression developmental changes in SMBC suggested that CAST mRNA expression in different tissues has obviously development change in SMBC.CAST mRNA expression in muscle of 42th day were significantly higher than other weeks.Moreover,CAST mRNA were extensively present Tibetan chicken at age of one day old and Daheng qualitity meat chicken at age of 10 weeks old.CAST mRNA high expressed in muscle tissues in different species.