Cloning, Expressing And Polymorphism Analysis Of SRPK1 And SRPK3 In Pig

Arginine-serine protein kinase (SRPK) is a family of the SR protein kinase-specific phosphorylation. Some studies show that SRPK1 is located in a control fat traits QTL region of fat, the high or low of SRPK3 while lead to muscle satellite cells lesions or lead to death of individual organisms. Therefore, the study of molecular genetic structure and spatial-temporal expression with modern molecular biology to research pig SRPK1, SRPK3 gene,a candidate gene for meat quality traits are very important in theoretical significance.In this study, Yorkshire, Duroc, northeast wild boar, northeast China pigs, Crossbred boars as the research object. Cloning of the SRPK1 gene and SRPK3 genes, analyzed its structure and function in molecular evolution. And comparative analysis of the situation of the heart, liver, spleen, stomach, kidney, lung, large intestine, small intestine, brain, muscle expression of the one month old Yorkshire and Duroc, and 1-day-old Yorkshire. The polymorphism of some exons in SRPK1 and SRPK3 gene was examined. To build pig skeletal muscle injury model to detect the expression of SRPK1, SRPK3 in the process of of the skeletal muscle injury and repair. And through the model to built the interaction networks of SRPK1, SRPK3, MyoD, FoxOI, MEF2A, MEF2B and MEF2D gene. The major findings as follows:(1) To Yorkshire pigs as the study object, using RT-PCR cloning long 2499bp Yorkshire SRPK1 gene sequences, covered 1968bp complete CDS region, which includes the use of inverse-PCR cloning of pigs SRPK1 part of the 5'UTR. Result showed that the encoded protein contains 656 amino acids; get SRPK3 gene 1708bp nucleotide fragments, covered full-length gene CDS(1701bp)of SRPK3.(2) Use the bioinformatics to analysis nucleic acid sequences and structural features of protein-coding sequences of SRPK1 and SRPK3 found that each includes a P_Kc and an ATP-binding domain, pigs SRPK1 protein sequence has high similarity with human and chimpanze. Through the bioinformatics prediction found the part of the 5'UTR of SRPK1 contains multiple transcription binding sites:HSF1, HSF2, Ik-1, IK-2, SRY, SP1 MyoD, USF, E47, p300, CP2, RREB-1, E2F, AP-1. Pig SRPK3 protein sequence has high similarity with human and bovine.(3) Used PCR-SSCP technique to analysis the 8th,10th,11th,16th exons of SRPK1 gene and 6th,9th exons of SRPK3 on Yorkshire, Northeast Min pigs, Northeast and Crossbred boars. The results showed that there was not any SNPs in exons region of SRPK1 tested, but found mutation at sixth exon of SRPK3 as A629→G629, T653→T653, amino acid changes for the Pro→His, Ile→Thr; the nineth exon mutation as G1059→A1059, amino acid no mutation.(4) Use the Realtime-PCR showed that the expression of pig SRPK1 and SRPK3 gene both have organizational and species-specific, but SRPK1 mostly located in the stomach, large intestine and small intestine and other parts of the digestive system and nutrition absorption-related. And the SRPK3 mainly expressed in muscle and heart.(5) Construction of pig skeletal muscle injury model combined with the Realtime technology to detect the expression of SRPK1 and SRPK3 showed that SRPK1 gene in the expression of injury and repair process and reduce the recurring increase, SRPK3 expression continues to rise.(6) The study also examined, including MyoD, MEF2A, including several other myogenic factors in skeletal muscle count in the expression of the repair process, and to build a cross doing related research results showed that only SRPK1 associated with SRPK3 significant (q< 0.01), SRPK1 associated with MEF2D significant (q< 0.05), other groups are not significant (q> 0.05).