Molecular Cloning,Characterization And Tissue Expression Analysis Of The Ovine CSRP2、CSRP3 And FSTL1 Genes From Small-tail Han Sheep

According to the Small tail Han sheep and Dorper sheep muscle transcriptome sequencing data, selecting the differentially expressed genes-CSRP2, CSRP3 and FSTL1 as our target genes. CSRP2 plays important roles in vascular smooth muscle cell growth, development and structure maintenance. CSRP3 is specifically expressed in striated muscle, and plays an important role in the process of striated muscle cell growth and development; studies have shown that CSRP3 is a positive regulator of muscle differentiation. FSTL1 plays roles in the process of fat cell growth and development. Studies have suggested that down regulation of FSTL1 expression may be an important feature of preadipocyte to adipocyte conversion. However, few CSRP2, CSRP3 and FSTL1 genes have been functionally characterized in sheep.In this study, the full-length cDNAs of the CSRP2, CSRP3 and FSTL1 genes were cloned from Small-tail Han sheep by rapid amplification of cDNA ends-PCR. Structure, characteristics of c DNA and protein sequences was analyzed by bioinformatics software. mRNA expressing profile of these genes were analyzed by qRT-PCR. The expression level of CSRP3 encoding protein was analyzed by western blotting. The main results were showed as follows:(1) The GenBank accession numbers of the full-length CSRP2, CSRP3 and FSTL1 cDNA sequences are KJ743957,KJ743958 and KJ872498, respectively. The complete ovine CSRP2 cDNA is 917 bp long and encoding 193 amino acids. The complete ovine CSRP3 cDNA is 917 bp long and encoding 194 amino acids. The complete ovine FSTL1 cDNA is 3634 bp long and encoding 307 amino acids. The ovine CSRP2 and CSRP3 genes include 6 exons and 5 introns; whereas, FSTL1 includes 11 exons and 10 introns.(2) The isoelectric points(pI) of CRP2 and CRP3 were estimated at nearly 9, respectively. CRP2 and CRP3 were predicted to contain one or more N/O-glycosylation sites and phosphorylation sites. Over 80 percent of their secondary structures were Coil. 1B8 T was the best tertiary structure template for CRP2 and CRP3, which contained two LIM domains and a nuclear targeting sequence. FSTL1, acidic protein, had a signal peptide sequence(1-19aa). FSTL1 was predicted to possess four O-glycosylation sites and 19 phosphorylation sites. Nearly 65 percent of their secondary structures were Coil. 1BMO was the best tertiary structure template for FSTL1, which contained FOLN domain, Kazal domain, EF-hand domain and VWFC domain.(3) Alignment analyses revealed that the CRP2, CRP3 and FSTL1 amino acid sequences are highly similar to those of other vertebrates. According to clustering results, sheep was most similar with cattle and goat, while zebrafish and xenopus laevis were far from above vertebrates. Phylogenetic analyses revealed that the CRP2 and CRP3 were closer relations in lower vertebrates(like fish) than those in higher vertebrates(like mammals). Two proteins were ortholog clusters, suggesting that these genes have evolved before species differentiation.(4) CSRP2 was mainly detected in the aorta, whereas CSRP3 was highly concentrated in the heart and the muscle. CSRP3 was expressed to a higher level in the hearts of Small-tail Han sheep than in Dorper sheep(p<0.05). However, the opposite was found in the muscle(the longissimus dorsi and biceps femoris); CSRP3 was expressed to a higher level in Dorper sheep than in Small-tail Han sheep(p<0.05).The FSTL1 mRNA is mainly expressed in Perirenal fat, lung, aorta and subcutaneous fat, significantly higher than the expression in the liver, kidney, heart and longissimus dorsi(p< 0.05). In subcutaneous fat, the expression level of FSTL1 mRNA was significantly higher in Dorper than that in Small tail Han sheep(p<0.05).(5) CRP3 protein was mainly expressed in heart and skeletal muscle. We also detected a potentially novel isoform of the CSRP3 protein in sheep, which was significantly different in the heart tissue of the two breeds(p<0.05). The expression level of CRP3 protein was significantly higher in Dorper than that of Small Tail Han sheep(p<0.05). In these two breeds, the expression level of CRP3 protein was significantly higher than that of CRP3-b protein(p<0.05). In longissimus dorsi muscle, the expression level of CRP3 protein of small tail Han sheep was higher than that of Dorper(p<0.05). The results suggesting a vital role for the two isoforms in striated muscle cell development in sheep.In summary, this study lay the scientific foundation for revealing the genetic regulation mechanism of CSRP2, CSRP3 and FSTL1 genes of Small-tail Han sheep and Dorper sheep through the systematic study on the sheep CSRP2, CSRP3 and FSTL1 genes at different levels.