| The Endothelin family genes of sheep including EDNRA and EDNRB,EDNRs(Endothelin receptors)have been found to be widely expressed in the cardiovascular system,nervous system and gastrointestinal tract,it is closely related to cell proliferation and vascular contraction and relaxation,gastrointestinal motility and glandular secretion.At present,there is no report on the complete nucleotide sequence of EDNRA and EDNRB genes in sheep.In this experiment,the Small-tailed Han sheep and Dorper sheep were used as experim materials,cloned the full-length sequences of sheep EDNRA gene and EDNRB gene,to predict and analyze the sequence characteristics and the characteristics of the protein and detected its expression in Small-tailed Han sheep and Dorper sheep in multiple tissues.We used a lot of research methods such as RT-PCR,RACE,bioinformatics analysis,fluorescence quantitative PCR and other techniques.The main results are as follows:(1)The cDNA sequences of sheep EDNRA and EDNRB genes were cloned and analysised.The full length of EDNRA gene,including 7 exons,encoding 427 amino acids.The EDNRB gene is 4540 bp,containing 8 exons,encoding 41 amino acids.The full length of the gene has been submitted to GenBank,and the access number was KY659244,KY659245.(2)Physicochemical properties analysis showed that: both EDNRA and EDNRB were basic protein,very good in liquidity,unstable in structure.The kind of amino acid composition of the two proteins was the same and the proportion was similar.The two proteins had obvious trans-membrane domain,multiple glycosylation sites and phosphorylation sites,multiple protein binding sites and nucleotide binding sites.The signal peptide is obvious in these proteins..The main secondary structure of proteins were α-helix,the domain are both typical 7-TM domain.The template of third-structure of proteins were EDNRB from human.(3)The results of the amino acid sequence alignment showed that:the sequence similarity of the amino acid sequences of the two proteins was more than 80% in the selected species,and EDNRA was 97.28%.indicating that the two proteins are highly conserved in mammals,and EDNRA is more conservative and stable than EDNRB.(4)The highest expression of EDNRA gene is ovary in Small-tailed Han sheep and Dorper sheep,secondary is spleen and stomach.The expression in the ovary and stomach of the Small-tailed Han sheep were significantly higher than those of the Dorper sheep.The expression of EDNRB gene in Small-tailed Han sheep and Dorper sheep in the stomach,kidney and liver were relatively high.The expression of the EDNRB in Dorper sheep liver was significantly higher than small tail Han sheep(P<0.05).In two sheep,the expression of two genes with significant differences in the ovary,kidney(P<0.05).The expression of EDNRB gene was significantly higher than that of EDNRA in the liver in Dorper sheep(P<0.05).(5)The results of Western blot showed that the molecular weight of EDNRA protein was 63 kDa,which was the highest in liver.The Subtype EDNRB(EDNRB2)was found in the Western blot.Moreover,the expression of this subtype was significantly higher than that of EDNRB protein in different tissues.In summary,In this study,the Small-tailed Han sheep were used as experimental materials,and the EDNRA and EDNRB genes were cloned successfully.The characteristics of two genes and their expressed proteins were analyzed by bioinformatics software.The characteristics and functions of two genes were explored from the mRNA level and protein level.It is helpful To provide the basic theoretical basis for the subsequent study of gene function and molecular breeding of sheep. |
