小尾寒羊SPARCL1基因编码区克隆及表达分析

doi:10.16431/j.cnki.1671-7236.2019.12.002

Abstract

Abstract: This study was aimed to explore the gene structure of cysteine-rich acidic and rich in cysteine-like 1 (SPARCL1) and its expression differences in tissues of Small-tail Han sheep.The total RNA was extracted from the liver tissue of Small-tail Han sheep.Primers were designed based on the sequence of SPARCL1 gene in sheep published in GenBank,and the SPARCL1 gene codings region (CDS) was amplified by PCR.The amplified product was constructed into pMD18-T vector for sequencing,and the sequence information of the complete CDS of SPARCL1 gene was obtained.The sequence and protein structure were analyzed by bioinformatics software.The expression of SPARCL1 gene mRNA in different tissues (liver,muscle,stomach,duodenum and small intestine) of Small-tail Han sheep was detected by Real-time quantitative PCR.The results showed that the length of SPARCL1 gene CDS was 1 962 bp,encoding 653 amino acids;The molecular weight was 74.39 ku,the theoretical isoelectric point was 4.64,which was a hydrophilic protein;SPARCL1 gene had a information peptide cleavage site and it was a secreted protein;The homology of SPARCL1 gene sequence in Small-tail Han sheep was 99.80% with that of sheep provided by NCBI,and there were 4 mutation sites of SPARCL1 gene CDS,but no amino acid changes were caused;There were 77 protein phosphorylation sites and 3 glycosylation sites of SPARCL1 protein.SPARCL1 protein expression was mainly localized in cytoplasm.The alpha helix,beta fold,beta turn and random coil of SPARCL1 protein in the secondary structure were 31.9%,6.4%,28.7% and 33.0%,respectively,the tertiary structure prediction result of SPARCL1 protein were consistent with the secondary structure.The relative expression of SPARCL1 gene in subcutaneous adipose tissue of Small-tail Han sheep was significantly higher than that in other tissues.In this study,the complete CDS sequence of SPARCL1 gene was successfully cloned,and its sequence components,protein physicochemical properties,structure and expression differences among tissues were analyzed,which provided a theoretical basis for studying the function of SPARCL1 gene and exploring its possible role of fat metabolism in Small-tail Han sheep.

Key words: Small-tail Han sheep; SPARCL1 gene; coding region; cloning; tissue; expression difference

CLC Number:

XIAO Cheng, JIN Haiguo, WEI Tian, GAO Yi, YU Yongsheng, ZHANG Lichun, MA Huihai, CAO Yang. Cloning and Expression Analysis of SPARCL1 Gene Coding Region in Small-tail Han Sheep[J]. China Animal Husbandry and Veterinary Medicine, 2019, 46(12): 3466-3474.

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Cloning and Expression Analysis of SPARCL1 Gene Coding Region in Small-tail Han Sheep

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