Molecular Cloning And Characterization And Expression Analysis Of ANKRD2 Gene In The Small-tailed Han Sheep

The family of muscle specific ankyrin repeat proteins(MARPs) includes cardiac ankyrin repeat protein(ANKRD1), Ankyrin repeat domain protein 2(ANKRD2), and diabetes related ankyrin repeat protein(ANKRD23). According to the Small-tailed Han sheep(SH) and Dorper sheep(DP) muscle transcriptome sequencing data, we selected the differentially expressed gene ANKRD2 as our target gene. ANKRD2 is specifically expressed in skeletal muscle, and plays important roles in the process of skeletal muscle growth and development,participate in muscle cell differentiation and the formation of the muscle fibers. However, ANKRD2 nucleic acid sequence, molecular structure and organization had not been reported in sheep. In this study, the full-length c DNA of ANKRD2 gene was cloned, a series of bioinformatics analysis of the gene and its encoding protein was conducted, and the expression abundance of this gene at mRNA and protein levels in SH and DP. The main results were showed as follows:(1) The complete cDNA sequence of the Small-tailed Han sheep ANKRD2 gene was cloned using RT-PCR, 5? RACE and 3? RACE, which was 1179 bp in length, including 990 bp open reading frame, 21 bp 5 ' UTR and 168 bp 3' UTR. The open reading frame encodes 329 amino acid polypeptide chain. The GenBank accession numbers of the full-length cDNA sequences that we cloned was KR090892. We compare the cloned sequence with the predicted sequence, found that 99.40% accuracy, there are existing two base mutations in the CDS area.(2) Bioinformatics analysis showed that ANKRD2 gene encoding protein molecular weight was 36.7 kDa. The Aliphatic index was 87.48(>60), showed that the protein had a bigger liquidity. The instability index was 49.57(>40), showed that the protein had a generally stability. The hydrophobic index was-0.6, showed that the protein was a water- soluble protein, and had a good hydrophilicity. The isoelectric point index was 5.72, showed that it is acidic protein, and the most composition of amino acid was glutamic acid. The secondary structure of the protein was predicted to be ?-helical and irregular curl. The C value, S value and Y values are less than 0.5, concluded that there is no signal peptide protein, not belongs to a transmembrane protein, it cannot be secreted to the outside of the cells play a role. In the amino acid sequence, fourteen phosphorylation sites, twelve ubiquitination sites, ten glycosylation sites and four anchor structure domain. Using the Swiss-model software forecast that model ANK-N5C-317(4o60.1.A) is the most similar in tertiary structure.(3) Homology analysis with other species of amino acid shows that Small-tailed Han sheep ANKRD2 protein and selected species were over 86% similarity, indicated that the protein in mammals highly conservative, similar to goats, cows, horses, and human, swine, rat, whales are far apart.(4) The expression of the gene was evaluated in various SH and DP tissues using qRT-PCR and Western-blot analyses. The results showed clear tissue specificity in both sheep breeds, and was primarily expressed in the skeletal muscle. the expression levels in the skeletal muscle were significantly different from those in the other tissues(p<0.05);. Furthermore, in the skeletal muscle, the expression of ANKRD2 protein in DP was significantly higher than that in SH(p<0.05). However, the expression of ANKRD2 in the other tissues was similar between DP and SH.In summary, this study from the molecular level lays the scientific foundation for revealing the genetic regulation mechanism of ANKRD2 gene of Small-tailed Han sheep and Dorper sheep through the systematic study on the sheep ANKRD2 gene, and verified the ANKRD2 gene in the Small-tailed Han sheep and Dorper sheep expression in different organizations.