<em>CIDEA</em> 基因对奶牛乳腺上皮细胞脂代谢相关基因表达及甘油三酯合成的影响

doi:10.3969/j.issn.1674-7968.2023.05.008

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摘要诱导细胞死亡的 DNA 片段化因子-α 样效应物 A (cell death-inducing DNA fragmentation factor α- like effector A, CIDEA)是一种脂滴(lipid droplet, LD)相关蛋白,在哺乳期的乳腺中高度表达,介导小脂滴变成大脂滴并促进甘油三酯(triglyceride, TAG) 的积累。为了检测CIDEA 基因对奶牛乳腺上皮细胞(bovine mammary epithelial cells, BMEC)中脂代谢相关基因表达及 TAG 合成的影响,本研究通过 qPCR 技术检测奶牛(Bos taurus)干奶期和泌乳盛期乳腺组织中的 CIDEA 基因表达变化;设计合成靶向 CIDEA 基因的siRNA检测脂代谢相关基因的表达;利用 TAG 试剂盒检测细胞内 TAG 的含量。结果表明,通过克隆得到全长 660 bp 的奶牛 CIDEA 基因(GenBank No. MW960011)的 CDS 区序列; 对其进行了生物信息学分析发现,奶牛 CIDEA 基因核苷酸序列与婆罗门牛(B. indicus)(GenBank No. XM_019986952.1)、牦牛(B. mutus) (GenBank No. XM_005892367.1)、印度水牛(Bubalus bubalis)(GenBank No. XM_006043025.3)的同源性分别为 99.73%、99.46% 和 98.52%,氨基酸序列同源性均在 95% 以上;qPCR 检测发现,CIDEA 基因在奶牛泌乳盛期的表达量是干奶期的 1.5 倍;在 BMEC 中转染 siRNA,干扰效率达到 95% 以上;干扰 CIDEA 基因后, 显著下调了脂肪酸从头合成相关基因固醇调节元件结合蛋白1(sterol-regulatory element binding proteins 1, SREBF1)、乙酰辅酶 A 羧化酶 α (acetyl-CoA carboxylaseα, ACACA)的表达, 同时抑制了 TAG 合成相关基因二酰甘油酰基转移酶1(diacylglycerol O-acyltransferase 1, DGAT1)、DGAT2、脂素1(lipin 1, LPIN1)和脂肪酸转运相关基因心脏脂肪酸结合蛋白3(heart fatty acid binding protein 3, FABP3)、人类白细胞分化抗原36 (cluster of differentiation 36, CD36) 的表达 (P<0.05),并且促进了甘油三酯水解酶(adipose triglyceridelipase, ATGL)的表达(P<0.05);CIDEA 干扰后, BMEC 内 TAG 含量显著下降(P<0.05)。综上所述, CIDEA 基因在调节 BMEC 脂代谢相关基因及TAG 合成方面发挥着重要的作用。本研究进一步明确乳脂代谢的调控机理,对奶牛产业的健康发展具有重要的理论和实践意义。

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关键词 ：奶牛, 乳腺上皮细胞(BMEC), 诱导细胞死亡的 DNA 片段化因子-α样效应物 A (CIDEA) , 乳脂代谢, 甘油三酯(TAG)

Abstract：CIDEA is a lipid droplet (LD) -associated protein that is highly expressed in the lactating mammary gland, mediates the transformation of small lipid droplets into large lipid droplets and promotes triglyceride (TAG) accumulation. In order to detect the effect of CIDEA gene on lipid metabolism-related gene expression and TAG synthesis in mammary epithelial cells of dairy cows (Bos taurus), the expression of CIDEA gene in mammary tissues of dairy cows during dry milk and lactation was detected by qPCR technique; the synthesis of siRNA targeting CIDEA gene was designed to detect the expression of genes related to lipid metabolism; and the intracellular TAG content was detected by TAG kit. Results showed that the sequence of the CDS region of the full-length 660 bp cow CIDEA gene (GenBank No. MW960011) was obtained by cloning. Bioinformatics analysis revealed that the nucleotide sequences of B. indicus (GenBank No. XM_019986952.1), B. mutus (GenBank No. XM_005892367.1) and Bubalus bubalis (GenBank No. XM_006043025.3) were 99.73%, 99.46% and 98.52%, respectively, and the amino acid sequence homology was more than 95%. The qPCR technique revealed that the expression of CIDEA gene was 1.5 times higher in dairy cows during peak lactation than dry period; siRNA was transfected in dairy cows mammary epithelial cells and the interference efficiency reached over 95%. Interfering with the CIDEA gene significantly downregulated the expression of sterol regulatory element binding protein 1 (SREBF1) and acetyl coenzyme A carboxylase alpha (ACACA), and inhibited the expression of diacylglycerol acyltransferase 1 (DGAT1), DGAT2, lipin 1 (LPIN1), and heart fatty acid binding protein 3 (FABP3), cluster of differentiation 36 (CD36)(P<0.05), and promoted TAG hydrolase (ATGL) expression (P<0.05). After CIDEA interference, a significant decrease in intracellular TAG content was detected (P<0.05). In conclusion, CIDEA plays an important role in regulating genes related to lipid metabolism and TAG synthesis in BMEC. This study further clarifies the regulation mechanism of lipid metabolism in dairy cows, which has important theoretical and practical implications for the healthy development of the dairy industry.

Key words： Dairy cows Mammary epithelial cells (BMEC) Cell death-inducing DNA fragmentation factor α-like effector A (CIDEA) Milk lipid metabolism Triglycerides (TAG)

收稿日期: 2022-05-23

ZTFLH:

S823

基金资助:国家自然科学基金(31702095); 天津市农业科学院青年科研人员创新研究与实验项目(2021010); 天津市科技计划项目(19ZXZYSN00020); 种业创新研究项目(2022ZYCX010); 天津市种业科技重大专项(19ZXZYSN00130); 天津市科技计划项目(22ZXZYSN00020)

通讯作者: **chencb@nankai.edu.cn;tjnnyz@126.com

作者简介: *同等贡献作者

引用本文:

赵欣, 姚大为, 赵淑颖, 杨春蕾, 张漫, 陈成彬, 马毅. CIDEA 基因对奶牛乳腺上皮细胞脂代谢相关基因表达及甘油三酯合成的影响[J]. 农业生物技术学报, 2023, 31(5): 979-988.
ZHAO Xin, YAO Da-Wei, ZHAO Shu-Ying, YANG Chun-Lei, ZHANG Man, CHEN Cheng-Bin, MA Yi. Effects of CIDEA Gene on Lipid Metabolism Related Gene Expression and Triglyceride Synthesis in Dairy Cow (Bos taurus) Mammary Epithelial Cells. 农业生物技术学报, 2023, 31(5): 979-988.

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http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2023.05.008 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2023/V31/I5/979

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