Abstract: To investigate the regulation of alternative polyadenylation（APA） of the bovine secreted protein acidic and rich in cysteine（SPARC）gene in the proliferation and differentiation of preadipocytes，the existence of APA phenomenon of bovine SPARC gene was verified by 3' rapid amplification of cDNA ends（3' RACE）. Using dual luciferase reporter gene assay，qPCR，Western blotting and other molecular biology techniques，the differential expression characteristics of SPARC gene 3'untranslated regions（3'UTR）‑APA isoforms were analyzed.The reporter gene vector was set as wild‑type short 3'UTR group（WT‑3'UTRS） ， wild‑type long 3'UTR group （WT‑3'UTRL） ， mutant long 3'UTR group（Mut‑3'UTRL‑P1），respectively. The protein expression vector was set as wild‑type short 3'UTR fusion group WT‑SPARCS），wild‑type long 3'UTR fusion group（WT‑SPARCL），and mutant long 3'UTR fusion group Mut‑SPARCL‑P1）. Through functional gain and loss test，CCK‑8 proliferation test，oil red O staining and qRT‑PCR technology，SPARC overexpression group（pCMV‑SPARC），empty vector control group（pCMV），SPARC interference group（siSPARC）and negative control group（NC）were set up to systematically explore the effects of SPARC gene and its 3'UTR‑APA isoforms on the proliferation and adipogenic differentiation of bovine preadipocytes. The results showed that there were two 3'UTR‑APA isoforms in bovine SPARC gene. Compared with the WT‑3'UTRS group，the luciferase activities of the WT‑3'UTRL and Mut‑3'UTRL‑P1 groups were significantly or extremely significantly decreased.Compared with the WT‑SPARCS group，the mRNA and protein expression levels of the SPARC gene in the WT‑SPARCL and Mut‑SPARCL‑P1 groups were both highly significantly downregulated. Compared with the pCMV group，the mRNA expression levels of proliferation marker genes in the pCMV‑SPARC group were extremely significantly upregulated，which significantly promoted cell proliferation，while the mRNA expression levels of adipogenic marker genes were extremely significantly downregulated，and lipid droplet accumulation was extremely significantly reduced. Compared with the NC group，the mRNA expression level of proliferation marker genes in the siSPARC group showed the opposite trend. Compared with the WT‑SPARCL group，the mRNA expression levels of proliferation marker genes in the WT‑SPARCS group were generally significantly or highly significantly upregulated，and cell proliferation was significantly promoted；meanwhile，the mRNA expression levels of adipogenic marker genes were highly significantly down‑regulated，and lipid droplet accumulation was significantly reduced.In conclusion，the functional differences of the APA regulation mechanism of bovine SPARC gene in the proliferation and differentiation of its preadipocytes were revealed，which not only provided a theoretical reference for analyzing the molecular mechanism of its participation in the post‑transcriptional regulation of bovine fat deposition，but also laid a theoretical foundation for molecular breeding of high‑quality beef cattle.

Key words: Bovine SPARC gene, Alternative polyadenylation, Cell proliferation, Adipogenesis differentiation

摘要： 为探究牛富含半胱氨酸的酸性分泌蛋白（SPARC）基因的可选择性多聚腺苷酸化（Alternative polyadenylation，APA）对前脂肪细胞增殖与分化的调控作用，通过cDNA 3'末端快速扩增技术验证牛SPARC基因APA现象的存在；利用双荧光素酶报告基因试验、qPCR、Western blotting等分子生物学技术，分析了SPARC基因3'非翻译区（3'UTR）-APA异构体的差异表达特征，其中报告基因载体分别设置为野生型短3'UTR 组（WT-3'UTRS）、野生型长3'UTR 组（WT-3'UTRL）、突变型长3'UTR 组（Mut-3'UTRL-P1），蛋白质表达载体设置为野生型短3'UTR融合组（WT-SPARCS）、野生型长3'UTR融合组（WT-SPARCL）、突变型长3'UTR融合组（Mut-SPARCL-P1）；通过功能获得与缺失试验、CCK-8增殖试验、油红O染色、qRT-PCR技术，设置SPARC过表达组（pCMV-SPARC）、空载体对照组（pCMV）、SPARC干扰组（siSPARC）、阴性对照组（NC），系统探究SPARC 基因及其3'UTR-APA异构体对牛前脂肪细胞增殖与成脂分化的影响。结果表明，牛SPARC 基因中存在2种3'UTR-APA异构体；与WT-3'UTRS组相比，WT-3'UTRL及Mut-3'UTRL-P1组的荧光素酶活性显著或极显著降低；与WT-SPARCS组相比，WTSPARCL及Mut-SPARCL-P1组的SPARC 基因mRNA及蛋白质表达量均极显著下调。与pCMV组相比，pCMV-SPARC 组的增殖标志基因mRNA表达量极显著上调，极显著促进细胞增殖，而成脂标志基因mRNA表达量均极显著下调，脂滴积累量极显著降低；与NC组相比，siSPARC组的增殖标志基因mRNA表达情况则相反；与WT-SPARCL组相比，WT-SPARCS组的增殖标志基因mRNA表达量总体上显著或极显著上调，显著促进细胞增殖，成脂标志基因mRNA表达水平极显著下调，脂滴积累量显著降低。综上，研究揭示了牛SPARC基因的APA调控机制在其前脂肪细胞增殖与分化中的功能差异，不仅为解析其参与牛脂肪沉积转录后调控的分子机制提供了理论参考，也为优质肉质牛的分子育种奠定了理论基础。

关键词: 牛SPARC基因, 可选择性多聚腺苷酸化, 细胞增殖, 成脂分化