Cloning, Expression Patterns And Genetic Variation Of Insigs Gene In Qinchuan Beef Cattle

There are two members in insulin induced gene family: INSIG1and INSIG2, both ofthem involved in lipogenesis, cholesterol metabolism and the regulation of energy and bodyweight. In this experiment, we take both INSIG1and INSIG2as candidate gene to study thefunction of them in Qinchuan beef cattle. In this study, we collected the liver samples ofQinchuan cattle to clone the INSIG2gene, using bioinformatics methods to predict thestructure and function of this gene. Analyzed the expression pattern in different tissues ofQinchuan cattle by real-time fluorescent quantitative PCR. Used PCR-RFLP, PCR-SSCP andDNA sequencing to detect the SNP of the two genes in Qinchuan cattle and their associationwith the growth and carcass traits. The main results were showed as following:1. Cloning and bioinformatics analysis of the INSIG2gene in Qinchuan cattleThe362bp of INSIG2gene was cloned by RT-PCR, including completely CDS region.We submitted it to GenBank and obtained the accession number of the sequence(JQ951942).The ORF of INSIG2gene including351bp nucleotides, encoding116amino acids. The relativemolecular mass and PI of INSIG2protein are12.8kD and7.73, respectively. The maximum hydrophobicityvalue is2.444and minimum value is-1.533, located at54th,55th and40th amino acids. Three strongtransmembrane helices located at22-39aa,45-65aa and76-97aa. Three phosphorylation sites are located at16th,44th and78th amino acids. No signal peptide domain was found in INSIG2protein.2. Expression pattern of the INSIG1and INSIG2genes in Qinchuan cattleExpression pattern of INSIG1and INSIG2genes in liver, luang, spleen, stomach, heart,abdominalfat, muscle and intestine of Qinchuan cattle by real-time fluorescent quantitativePCR. The results showed that the expression of INSIG1mRNA was highest in liver and alllower levels in other seven tissues. For INSIG2gene, the expression of liver was the highestand then lungs, the expression of the other6tissues were all lower than the two tissues.3. Effects of polymorphisms of INSIG1gene on growth and carcass traits of bovineWe used the PCR-RFLP and DNA sequencing to detect the SNPs in INSIG1gene of21520±2month age Qinchuan cattle, and the association between the polymorphisms and growthand carcass traits were analyzed. A total of4SNPs were found, they were A4366G, T4534C,T5001C and A5235G, respectively. Genetic diversity results showed all of them were mediumpolymorphism. The associations between the mutation at locus A4366G with the growth andcarcass traits showed that genotype had a significant effect on hip width, slaughter weight and carcass weight(P<0.05). The genotypes at locus T4534C had a significant effect on bodylength, wither height and hip width(P<0.05).. The genotypes at locus T5001C had asignificant effect on hip width(P<0.05).. In addition, there were no significant differencesbetween genotypes at locus A5235G(P>0.05).4. Effects of polymorphisms of INSIG2gene on growth and carcass traits of bovinePCR-SSCP and DNA sequencing were used to detect the SNPs in INSIG12gene of21520±2month age Qinchuan cattle. A SNP of T15570C was found. Genetic diversity resultsshowed it was medium polymorphism. The association study showed that TT genotype hadsignificant taller body height than TC genotype(P<0.05), there are no significant differencesin other traits(P>0.05).In conclusion, we successfully cloned the INSIG2gene of Qinchuan cattle, it is usefulfor the further research on the function of this gene in the growth process of Qinchuan cattle.The expression patterns of INSIG1and INSIG2genes analysis provide a scientific theory onthe expression of relative traits. The purpose of this study is to settle the theory foundationson the native breeding work of Qinchuan beef cattle.