Cloning, Expression And Polymorphism Analysis Of ACSL4 In Wild Boar And Porcine

The content of fat is an important target and is the focus of the work to improve pig which the active of rate-limiting enzyme from the process of the fatty synthesis and catabolism has an effect on fatty deposition.Acly-CoA synthetase long chain family 4 (ACSL4) plays an important role in fatty acid metabolism. There are two steps to catalyze free fatty acid and coenzyme A.Acly-CoA synthetase is made when ATP and Mg2+ exist, and then participate in fatty acid synthesis and catabolism.This study using comparative genomics and bioinformatics analyzed constructional and evolutionary of ACSL4 gene of the coding region;using RT-PCR methods analyzed the ACSL4 gene's expressing quantitative in tissue;using PCR-SSCP methods analyzed polymorphism of pig breeds. The main results are as follows:1. The full-length coding region sequence of Wild boar ACSL4 gene (GenBank accession No:EU553523)was cloned and Yorkshire ACSL4 and partial sequence for the first time(GenBank accession No:EF641277),there were three differences in the coding region of Wild boar and Yorkshire,located (A1015G,T1468C,T1469C);there were two amino acids differences in the level of coding amino acids,located (R339G,F490P).From the bioinformatics analysis,there was a potential AMP binding site located 234-245 in polypeptide chain,which showed ACSL4 gene is controled by AMP binding protein. The ACSL4 gene molecular evolution phylogenetic tree was constructed by the method of adjunction, there was high homology in swine and cattle, the variation of fowl is wide,located the external phylogenetic tree.The result of genome structural analysis showed there were 14 exons and 13 introns in ACSL4 gene.2. The ACSL4 gene's organizational expressing spectra of Wild boar,Yorkshire and Cross breed was constructed by RT-PCR.The result shows that ACSL4 gene was expressed in muscle, uterus,kidney,lung and heart,was hardly expressed in stomach,large intestine,small intestine and liver;the analysis of the ACSL4 gene's expressing in the same tissue of Wild boar and Cross breed showed,in kidney,spleen,lung,heart,uterus and muscle,Yorkshire had more than Cross breed.3. The PCR-SSCP analysis showed,there were 4 point mutation in extron 8,11 and 14 in ACSL4 gene,which caused the change of amino acids in polypeptide chain R339G,F490P,G656W;it was detected that there were 2 mutable points in 3'UTR. Population genetics analysis, Detected by genotype frequency distributions of the polymorphic loci is associated with species.