奶牛SP1基因结构及对乳脂合成功能的初步分析

doi:10.11843/j.issn.0366-6964.2022.09.014

摘要/Abstract

摘要： 旨在探讨中国荷斯坦奶牛的特异性蛋白1（specificity protein，SP1）基因结构特征及其对奶牛乳脂合成的影响。根据NCBI已经公布的奶牛SP1基因序列（NM_001078027.1），利用生物信息学分析其序列保守性、理化性质、蛋白亲水性、蛋白质结构及互作蛋白；采用PCR技术扩增并克隆SP1基因CDS序列。然后，选取6头健康的中国荷斯坦奶牛，分别取泌乳期和干奶期奶牛乳腺组织，运用实时荧光定量PCR和Western blot方法检测SP1基因在不同时期奶牛乳腺组织中的表达情况。分离并纯化泌乳期奶牛乳腺上皮细胞，通过SP1过表达及干扰检测其对乳脂合成的影响，分别进行3次独立试验。结果显示，SP1基因序列在不同物种间高度保守，与山羊相似度最高（98.94%）。奶牛SP1基因CDS区序列长2 361 bp，编码786个氨基酸，蛋白分子质量为80 902.17 u，理论等电点为6.94。平均疏水指数为-0.438，为不稳定的亲水性蛋白。SP1序列包含3个锌指结构，SP1蛋白二级结构以无规则卷曲（52.29%）为主。STRING蛋白互作分析结果显示，SP1与转录因子AP-1（JUN）、雌激素受体α（ERα）、MYC原癌基因蛋白（MYC）、TATA盒结合蛋白（TBP）等蛋白存在相互作用。实时荧光定量PCR和Western blot结果显示，SP1的mRNA和蛋白在泌乳期的表达量显著高于干奶期（P<0.01）。在奶牛乳腺上皮细胞中过表达SP1显著增加细胞甘油三酯的合成（P<0.01），而干扰SP1的表达，甘油三酯合成显著降低（P<0.01）。以上结果提示，SP1正向调控乳脂合成，分析SP1基因结构和功能为深入研究SP1对泌乳奶牛乳脂合成调控机制提供理论依据。

关键词: 中国荷斯坦奶牛, SP1基因, 基因克隆, 生物信息学, 表达模式

Abstract: The study aimed to explore the structural characteristics of specificity protein 1 (SP1) and its effect on milk fat synthesis of Chinese Holstein dairy cows. According to the SP1 gene sequence of Bos taurus published by NCBI (NM_001078027.1), the SP1 sequence conservation, physical and chemical properties, protein hydrophilicity, protein structure and interaction proteins of the encoded protein were analyzed by bioinformatics softwares. The CDS region of SP1 gene was amplified and cloned by PCR. Then, 6 healthy Holstein dairy cows were used to study the effect of SP1 on milk fat synthesis. The mammary tissues of lactating cows and dry cows were collected, and the expression of SP1 in mammary tissue at different stages was detected by real-time quantitative PCR and Western blot. Next, the mammary epithelial cells were isolated and purified from the lactating cows and the effect of SP1 on milk fat synthesis were investigated by SP1 overexpression and RNAi. Each experiment was preformed triplicate. The results showed that the sequence of SP1 gene was highly conserved among different species, and had the highest similarity with goat (98.94%). The CDS region of SP1 gene in dairy cows was 2 361 bp, encoding 786 amino acids. The molecular weight of the protein was 80 902.17 u and the theoretical isoelectric point was 6.942. The average hydrophobic index of SP1 was -0.438, which was an unstable hydrophilic protein. The SP1 contained 3 zinc finger structures, and the secondary structure of SP1 protein was mainly random coil (52.29%). The results of STRING protein interaction analysis showed that SP1 interacted with transcription factor AP-1 (JUN), estrogen receptor α (ERα), MYC proto oncogene protein (MYC), TATA box binding protein (TBP) and other proteins. Real-time quantitative PCR and Western blot results showed that the mRNA and protein levels of SP1 in mammary tissues of lactating cows were significantly higher than those in dry cows (P<0.01). Overexpression of SP1 in mammary epithelial cells of dairy cows significantly increased the triglyceride content (P<0.01), while knockdown of SP1 expression significantly decreased the intracellular triglyceride content (P<0.01). These results suggest that SP1 positively regulate the milk fat synthesis. The study on the structure and function of SP1 gene provides a theoretical basis for further study on the regulatory mechanism of SP1 on milk fat synthesis in lactating dairy cows.

Key words: Chinese Holstein cows, SP1 gene, gene cloning, bioinformatics, expression pattern

中图分类号:

S823.9

杨洋, 周子薇, 张京一, 杨硕, 王博宇, 葛楠, 林叶, 侯晓明. 奶牛SP1基因结构及对乳脂合成功能的初步分析[J]. 畜牧兽医学报, 2022, 53(9): 2970-2981.

YANG Yang, ZHOU Ziwei, ZHANG Jingyi, YANG Shuo, WANG Boyu, GE Nan, LIN Ye, HOU Xiaoming. Analysis on SP1 Gene Structure and Its Function on Milk Fat Synthesis in Holstein Dairy Cows[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(9): 2970-2981.

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