草原红牛ACSL3基因CDS区克隆、生物信息学分析及组织表达研究

doi:10.16431/j.cnki.1671-7236.2019.04.001

Abstract

Abstract:

This study was aimed to clone the CDS of long-chain acyl-CoA synthetase 3 (ACSL3) gene in Red Steppe cattle,analyze it for bioinformatics,and detect the expression differences in different tissues of Red Steppe cattle at the mRNA and protein levels.The CDS of ACSL3 gene was obtained by RT-PCR and TA cloning methods.A variety of software and online tools were used to analyze the homology among different species and construct phylogenetic tree,analyze physical and chemical properties,potential phosphorylation locus,O-glycosylation sites,N-glycosylation sites,signal peptide,disulfide bonds,transmembrane region,subcellular localization,and the secondary and tertiary structures of ACSL3 protein.Meanwhile,the mRNA and protein expression levels of ACSL3 gene in each tissue of Red Steppe cattle were detected by Real-time PCR and Western blotting.The results showed that the CDS of ACSL3 gene was 2 163 bp in length encoding 720 amino acids with a protein molecular weight of 80.28 ku and a theoretical isoelectric point of 8.74,which was a hydrophilic protein.By NCBI BLAST comparison,the nucleotide sequence homology of ACSL3 gene in Red Steppe cattle shared 99%,97%,93%,91%,88%,88% and 78% identity with Bos taurus,Ovis aries,Sus scrofa,Homo sapiens,Mus musculus,Rattus norvegicus and Gallus gallus,respectively.The phylogenetic tree found that the Red Steppe cattle had the closest relationship with Bos taurus and Ovis aries,and the farthest relationship with Gallus gallus.The protein sequence had 7 disulfide bonds,66 phosphorylation sites,9 O-glycosylation sites,3 N-glycosylation sites,no signal peptide,but had a transmembrane region.The secondary and tertiary structures analysis showed that ACSL3 protein was connected by random coil,and the protein structure was mainly alpha helix and beta turn,which was a mixed protein.Real-time PCR and Western blotting results showed that the ACSL3 mRNA and protein were expressed in all the collected tissues,and the highest expression was found in kidney and muscle tissues,which were significantly higher than that in the other tissues (P<0.05);It was moderately expressed in stomach,liver and heart,which was significantly higher than that in spleen,lung,intestine and fat (P<0.05);While there were the lowest relative expression in spleen,lung,intestine and fat.The results showed that the differential expression levels of ACSL3 gene in Red Steppe cattle might be related to the regulation of fat deposition and lipid metabolism,which provided the basic materials for further study on the regulation of ACSL3 gene on lipid metabolism and fat deposition in Red Steppe cattle.

Key words: Red Steppe cattle; ACSL3 gene; clone; bioinformatics; differential expression; lipid metabolism

CLC Number:

LYU Yang, CAO Yang, GAO Yi, WANG Yuting, ZHANG Guoliang. Cloning,Bioinformatics and Tissue Expression Analysis of ACSL3 Gene CDS in Red Steppe Cattle[J]. , 2019, 46(4): 957-966.

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Cloning,Bioinformatics and Tissue Expression Analysis of ACSL3 Gene CDS in Red Steppe Cattle

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