小尾寒羊SP1基因克隆及其对前体脂肪细胞分化的影响

doi:10.16431/j.cnki.1671-7236.2021.05.005

Abstract

Abstract: The aim of this experiment was to clone the specificity protein 1 (SP1) gene,analyze its sequence,and investigate the effect of SP1 gene on the preadipocytes differentiation of Small-tailed Han sheep.The 1-month-old adipose tissue in the tail of Small-tailed Han sheep was selected for the isolation,culture and induction differentiation of preadipocytes.SP1 gene CDS was cloned by overlapping PCR.Bioinformatics softwares were used to analyze the SP1 gene CDS,and predict the structure,functional domain,subcellular localization and post-translational modification of SP1 protein.Overexpression and inhibition of SP1 gene in preadipocytes was performed by lentivirus infection.The overexpression and antisense vectors of SP1 gene and control plasmids packed with lentivirus were transfected into 293T cells,and the preadipocytes of Small-tailed Han sheep were infected with lentiviruses.Oil Red O staining was used to observe the ability of lipid accumulation in adipocytes.The mRNA expression of SP1 gene and adipogenic marker genes were detected by Real-time quantitative PCR.The results showed that the full-length of SP1 gene CDS was 2 340 bp,encoding 779 amino acids.Sequence similarity analysis showed that SP1 gene CDS of Small-tailed Han sheep had the highest similarity with Ovis aries and Capra hircas,followed by Bos taurus,Equus caballus,Sus scrofa,Homo sapiens,Mus musculus and Rattus norvegicus,and the lowest similarity with Gallus gallus,which was consistent with the result of phylogenetic analysis.The SP1 protein was located in the nucleus with 109 phosphorylation sites.Its secondary structure consisted of alpha helix,beta turn,radom coil and extended chain,and the proportion of them were 16.94%,8.60%,54.17% and 20.28%,respectively.Three zinc finger domains were found in both secondary and tertiary structures.The overexpression,interference and control plasmids were transfected into 293T cells,and the cells in each group presented strong green fluorescence,which indicated the vector was successfully transfected into 293T cells.After the lentiviruses infects the preadipocytes of the Small-tail Han sheep,the mRNA expression of SP1 gene in overexpression group was extremely significant higher than that in control group (P<0.01),while the expression of SP1 gene mRNA in inhibition group was extremely significant lower than that in control group (P<0.01).After SP1 gene was overexpressed,the mRNA expression of lipogenic marker genes were extremely significant up-regulated (P<0.01),and more lipid droplets were deposited.The results were opposite after SP1 gene was inhibited.Therefore,SP1 gene had a positive regulatory effect on the differentiation of preadipocyte in the caudal adipose tissue of Small-tailed Han sheep.

Key words: Small-tailed Han sheep; SP1 gene; cloning; preadipocyte; differentiation

CLC Number:

S811.3

WANG Feng, ZHAO Bishi, LIU Xuying, LIANG Yu, QIAO Liying, LIU Wenzhong, LIU Jianhua. Cloning of SP1 Gene and Its Effect on Preadipocytes Differentiation in Small-tailed Han Sheep[J]. China Animal Husbandry & Veterinary Medicine, 2021, 48(5): 1544-1557.

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Cloning of SP1 Gene and Its Effect on Preadipocytes Differentiation in Small-tailed Han Sheep

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