PDK4对山羊肌内脂肪细胞脂代谢的影响

doi:10.13560/j.cnki.biotech.bull.1985.2021-0368

摘要/Abstract

摘要：

旨在克隆获得山羊PDK4基因序列,明确其组织细胞表达特性,并明晰其对山羊肌内脂肪细胞脂代谢的作用。构建山羊PDK4过表达和干扰细胞模型,利用RT-PCR、实时荧光定量 PCR等研究PDK4对山羊肌内脂肪细胞脂代谢的影响。结果克隆获得山羊PDK4基因序列,长度为1 808 bp,定位于线粒体和细胞质。明确了山羊PDK4组织及细胞时序表达模式,发现PDK4高表达于山羊肺脏、臂三头肌和肝脏组织（P<0.01）;在诱导分化5 d的山羊肌内脂肪细胞中表达水平极显著高于诱导分化之前的表达水平（P<0.01）。干扰和过表达山羊PDK4分别显著降低和增加了脂质积聚,且干扰PDK4后脂代谢相关基因FABP3、CD36、ACACA、AGPAT6和ADRP表达水平均显著（P<0.05）或极显著（P<0.01）降低,而过表达PDK4后表达水平极显著升高（P<0.01）。PDK4在山羊各组织及肌内前体脂肪细胞分化各个阶段均存在表达。过表达山羊PDK4促进了脂肪细胞脂滴累积和脂质沉积相关基因表达上调,干扰后呈相反的趋势和表达。

关键词: 丙酮酸脱氢酶激酶4, 山羊, 表达特性, 脂代谢

Abstract:

This study aims to clone the goat PDK4 gene sequence,to clarify its tissue and temporal expression characteristics,and to clarify its effect on the lipid metabolism of goat intramuscular adipocytes. Overexpression and interference cell model of goat PDK4 was constructed,and RT-PCR and real-time fluorescent quantitative PCR were used to determine and study the effect of PDK4 on the lipid metabolism in goat intramuscular adipocytes. As results,the cloned goat PDK4 sequence was 1 808 bp,and it was located in mitochondria and cytoplasm. The expression pattern of goat PDK4 tissue and cell temporal expression was clarified,showing that PDK4 was highly expressed in the goat’s lung,triceps brachii muscle and liver tissue（P<0.01）. The expression level in goat intramuscular adipocytes at 5 d of induction was significantly higher than that before differentiation（P<0.01）. Interference and overexpression of goat PDK4 significantly reduced and increased lipid accumulation,respectively. The expression of lipid metabolism-related genes FABP3,CD36,ACACA,AGPAT6 and ADRP significantly（P<0.05）or extremely significantly（P<0.01）decreased with si-PDK4,while those genes extremely significantly increased（P<0.01）after overexpression of PDK4. In conclusion,PDK4 is expressed in goat tissues and in various stages of intramuscular preadipocyte differentiation. Overexpression of goat PDK4 promots the up-regulation of lipid droplet accumulation and lipid deposition-related gene expression in adipocytes,and shows the opposite trend and expression after interference.

Key words: pyruvate dehydrogenase kinase 4, goat, expression characteristics, lipid metabolism

张浩, 张亚楠, 李鑫, 王佳美, 王永, 朱江江, 熊燕, 林亚秋. PDK4对山羊肌内脂肪细胞脂代谢的影响[J]. 生物技术通报, 2021, 37(12): 151-159.

ZHANG Hao, ZHANG Ya-nan, LI Xin, WANG Jia-mei, WANG Yong, ZHU Jiang-jiang, XIONG Yan, LIN Ya-qiu. Effect of PDK4 on the Lipid Metabolism of Goat Intramuscular Adipocytes[J]. Biotechnology Bulletin, 2021, 37(12): 151-159.

图/表 7

表1 引物信息

Table 1 Primer information

基因 Gene	登录号 GenBank	退火温度 Annealing temperature/℃	序列 Primer sequence（5'-3'）	产物长度 Product length/bp	用途 Purpose
PDK4	NM_001101883.1	60	S:GAGTCTCAAGCCACCTACGG	1 806	PCR
PDK4	NM_001101883.1	60	A:TGACATCAATCAGTGGCCTAA	1 806	PCR
PDK4	MF564045.1	53	S:ACCTCTTTTGCATTTTTGCG	111	qPCR
PDK4	MF564045.1	53	A:TTTGGTCCTCTGGGCTTTTC	111	qPCR
TBP	XM_018053502.1	60	S:AACAGCCTCCCACCTTATGC	155	qPCR
TBP	XM_018053502.1	60	A:TGCTGCTCCTCCAAAATAGAC	155	qPCR
CD36	X91503	60	S:GTACAGATGCAGCCTCATTTCC	81	qPCR
CD36	X91503	60	A:TGGACCTGCAAATATCAGAGGA	81	qPCR
FABP3	NM_174313.2	58	S:GATGAGACCACGGCAGATG	120	qPCR
FABP3	NM_174313.2	58	A:GTCAACTATTTCCCGCACAAG	120	qPCR
AGPAT6	JI861797.1 XM_018041984.1	60	S:AAGCAAGTTGCCCATCCTCA	101	qPCR
AGPAT6	JI861797.1 XM_018041984.1	60	A:AAACTGTGGCTCCAATTTCGA	101	qPCR
ADRP	HQ846827 NM_001285596.1	60	S:TACGATGATACAGATGAATCCCAC	202	qPCR
ADRP	HQ846827 NM_001285596.1	60	A:CAGCATTGCGAAGCACAGAGT	202	qPCR
ATGL	GQ918145	60	S:GGAGCTTATCCAGGCCAATG	180	qPCR
ATGL	GQ918145	60	A:TGCGGGCAGATGTCACTCT	180	qPCR
UXT	NM_001037471	60	S:CAGCTGGCCAAATACCTTCAA	125	qPCR
UXT	NM_001037471	60	A:GTGTCTGGGACCACTGTGTCAA	125	qPCR

图1 山羊PDK4序列分析 A:PDK4序列及编码氨基酸信息;B:PDK4结构域预测;C:PDK4二级结构预测;D:PDK4互作蛋白预测

Fig. 1 Sequence analysis of goat PDK4 A:Information of PDK4 sequence and coding amino acid. B:Prediction of PDK4 domain. C:Prediction of PDK4 secondary structure. D:Prediction of PDK4 interaction protein

图2 山羊PDK4同源性分析（A）和进化树（B）

Fig. 2 Homology analysis（A）and phylogenetic tree（B）of goat PDK4

图3 山羊PDK4组织表达谱（A）和时序表达谱（B）

Fig. 3 Tissue expression profile（A）and temporal expression profile（B）of goat PDK4 *P<0.05,**P<0.01,The same below

图4 PDK4过表达（A）和干扰（B）效率

Fig. 4 Efficiency of PDK4 overexpression（A）and inter-ference（B）

图5 油红O染色及OD值

Fig. 5 Oil red O staining and OD value

图6 脂代谢相关基因表达水平

Fig. 6 Expression levels of lipid metabolism-related genes

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