干扰CREB基因对奶山羊乳腺上皮细胞脂质合成相关基因表达及甘油三酯合成的影响

doi:10.11843/j.issn.0366-6964.2019.12.005

畜牧兽医学报 ›› 2019, Vol. 50 ›› Issue (12): 2413-2421.doi: 10.11843/j.issn.0366-6964.2019.12.005

干扰CREB基因对奶山羊乳腺上皮细胞脂质合成相关基因表达及甘油三酯合成的影响

马静^1,2, 姚大为², 杨春蕾², 李秋玲³, 王添祯^1,2, 陈成彬^1*, 宋文芹¹, 马毅^2*

1. 南开大学生命科学学院, 天津 300071;
2. 天津市畜牧兽医研究所奶牛技术产业创新团队, 天津 300381;
3. 廊坊师范学院生命科学学院, 廊坊 065000

收稿日期:2019-05-20 出版日期:2019-12-23 发布日期:2019-12-20
通讯作者: 马毅,主要从事动物遗传育种与繁殖研究,E-mail:tjnnyz@126.com;陈成彬,主要从事分子细胞遗传学研究,E-mail:chencb@nankai.edu.cn
作者简介:马静(1994-),女,河北保定人,硕士生,主要从事动物遗传育种与繁殖研究,E-mail:15613563512@163.com;姚大为(1987-),女,河北涿州人,助理研究员,博士,主要从事动物遗传育种与繁殖研究,E-mail:yaodawei2008@126.com
基金资助:
国家自然科学基金（31702095）；天津市自然科学基金项目（18JCYBJC30200）

Effects of Interfering with CREB Gene on Lipid Synthesis Related Gene Expression and Triacylglycerol Synthesis in Mammary Epithelial Cells of Dairy Goats

MA Jing^1,2, YAO Dawei², YANG Chunlei², LI Qiuling³, WANG Tianzhen^1,2, CHEN Chengbin^1*, SONG Wenqin¹, MA Yi^2*

1. College of Life Sciences, Nankai University, Tianjin 300071, China;
2. Dairy Technology Industry Innovation Team, Tianjin Animal Husbandry and Veterinary Research Institute, Tianjin 300381, China;
3. College of Life Sciences, Langfang Normal College, Langfang 065000, China

Received:2019-05-20 Online:2019-12-23 Published:2019-12-20

摘要/Abstract

摘要： 旨在筛选奶山羊cAMP应答元件结合蛋白CREB（cAMP response element binding protein）基因的siRNA，揭示干扰该基因后对乳腺上皮细胞中乳脂合成相关基因表达及甘油三酯合成的影响。本研究通过qRT-PCR方法从西农萨能奶山羊乳腺组织中扩增CREB基因完整的CDS区，进行序列分析和不同泌乳时期表达水平分析，合成靶向CREB基因的siRNA，利用荧光定量PCR筛选有效siRNA，并检测脂质合成相关基因的表达，采用试剂盒检测细胞内甘油三酯含量。结果表明：1）克隆得到全长为984 bp的奶山羊CREB基因的CDS区（GenBank登录号：MK158073），并对其进行生物信息学分析。2）该基因在奶山羊泌乳盛期乳腺组织的表达量为干奶期的1.93倍（P<0.05）。3）成功筛选到靶向CREB基因的有效siRNA，干扰效率为72%（P<0.01）；并将其在奶山羊乳腺上皮细胞进行转染，通过qRT-PCR检测脂质合成相关基因的表达，与对照组相比，干扰CREB基因后显著抑制了FASN、ACACA、SCD1、FABP3、LPL、CPT1B、GPAM和DGAT2基因表达量（P<0.05），并显著增加了HSL基因表达量（P<0.05）；且细胞内甘油三酯含量被显著下调（P<0.05）。综上所述，CREB基因在奶山羊原代乳腺上皮细胞中很可能通过调控脂质代谢相关基因的表达及甘油三酯含量对山羊的乳脂合成过程发挥重要作用。

关键词: 奶山羊, 乳腺上皮细胞, CREB, RNA干扰, 脂质合成, 甘油三酯

Abstract: The study aimed to screen out the siRNA of cAMP response element binding protein (CREB) gene in dairy goats, and reveal the effect of interfering with CREB on the expression of milk fat synthesis related genes and triglyceride synthesis in mammary epithelial cells. The complete CDS region of CREB gene was amplified from the mammary gland tissue of Xinong Saanen dairy goat by qRT-PCR. Sequence and CREB expression analysis in different lactation periods were performed. The siRNA targeting the CREB gene was synthesized. The effective siRNA was screened by fluorescent quantitative PCR, at the same time, the expression of genes related to lipid synthesis was detected. The intracellular triglyceride content was detected using a kit. The results showed that:1) The CDS region of the CREB gene with 984 bp in dairy goat was cloned (GenBank accession number:MK158073) and analyzed by bioinformatics. 2) The expression level of CREB gene in the mammary gland of goats in lactating period was 1.93 times higher than that of goat in the dry period(P<0.05). 3) The effective siRNA targeting CREB gene was successfully screened, the interference efficiency was 72% (P<0.01),meanwhile transfecting it into the mammary gland epithelial cells of dairy goats. The expression of lipid synthesis-related genes were detected by qRT-PCR. Compared with the control group, the expression of FASN, ACACA, SCD1, FABP3,LPL, CPT1B,GPAM and DGAT2 genes was significantly inhibited after interfering with CREB gene (P<0.05), and the expression level of HSL was significantly increased(P<0.05),as well as the content of intracellular triglyceride was significantly inhibited(P<0.05). In summary, CREB gene maybe play an important role in goat milk fat synthesis by regulating the expression of lipid metabolism related genes and the content of triglyceride in primary mammary epithelial cells of dairy goats.

Key words: milk goat, mammary epithelial cell, CREB, RNA interference, lipid synthesis, triglyceride

中图分类号:

S287
S813.3

马静, 姚大为, 杨春蕾, 李秋玲, 王添祯, 陈成彬, 宋文芹, 马毅. 干扰CREB基因对奶山羊乳腺上皮细胞脂质合成相关基因表达及甘油三酯合成的影响[J]. 畜牧兽医学报, 2019, 50(12): 2413-2421.

MA Jing, YAO Dawei, YANG Chunlei, LI Qiuling, WANG Tianzhen, CHEN Chengbin, SONG Wenqin, MA Yi. Effects of Interfering with CREB Gene on Lipid Synthesis Related Gene Expression and Triacylglycerol Synthesis in Mammary Epithelial Cells of Dairy Goats[J]. Acta Veterinaria et Zootechnica Sinica, 2019, 50(12): 2413-2421.

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干扰CREB基因对奶山羊乳腺上皮细胞脂质合成相关基因表达及甘油三酯合成的影响

Effects of Interfering with CREB Gene on Lipid Synthesis Related Gene Expression and Triacylglycerol Synthesis in Mammary Epithelial Cells of Dairy Goats

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