小尾寒羊SP1基因克隆及其对前体脂肪细胞分化的影响

试验旨在对小尾寒羊特异性蛋白1（specificity protein 1，SP1）基因进行克隆和序列分析，并研究SP1基因对前体脂肪细胞分化的影响。选取1月龄小尾寒羊尾部脂肪组织进行前体脂肪细胞的分离、培养和诱导分化；用重叠PCR法克隆SP1基因编码区（coding DNA sequence，CDS）；用生物信息学软件分析SP1基因CDS，并对其编码蛋白的结构、功能结构域、亚细胞定位、翻译后修饰进行预测；用慢病毒包装SP1基因过表达、干扰及对照质粒转染293T细胞后，收取病毒液感染小尾寒羊前体脂肪细胞；用油红O染色检测脂滴沉积能力；用实时荧光定量PCR检测SP1基因和成脂标志基因的mRNA表达量。结果表明，小尾寒羊SP1基因CDS全长2 340 bp，编码779个氨基酸；序列相似性分析显示，小尾寒羊SP1基因CDS及其编码序列与绵羊、山羊的相似性最高，其次是牛、马、猪、人、小鼠、大鼠，与鸡的相似性最低，系统进化分析结果与之相符；SP1蛋白定位于细胞核，有109个磷酸化位点，其二级结构由α-螺旋、β-转角、无规则卷曲和延伸链4种结构组成，所占比例分别为16.94%、8.60%、54.17%和20.28%，二级结构和三级结构均存在3个锌指结构域；过表达、干扰及对照质粒转染293T细胞，每组细胞皆出现较强绿色荧光，载体成功转染至293T细胞；病毒液感染小尾寒羊前体脂肪细胞并分化12 d后，过表达组SP1基因表达量极显著高于对照组（P<0.01），干扰组SP1基因表达量极显著低于对照组（P<0.01）；过表达SP1基因极显著上调成脂标志基因mRNA的表达量（P<0.01），产生更多脂滴；干扰SP1基因则结果相反。因此，SP1基因对小尾寒羊尾部前体脂肪细胞分化具有正向调控的作用。

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

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.