Krüppel-Like Factor 3（KLF3）基因对牛脂肪沉积的作用

doi:10.3864/j.issn.0578-1752.2019.07.014

摘要/Abstract

摘要：

【目的】通过研究牛KLF3基因对牛脂肪分化和脂肪酸代谢关键基因表达量的作用,进而探讨KLF3 基因对脂质沉积的影响。【方法】合成干扰siRNA,筛选得到KLF3基因干扰效率最高SiRNA,分离培养秦川牛肉用新品系新生牛前脂肪细胞生长至70%-90%汇合度时转染筛选得到的KLF3基因SiRNA和阴性对照SiRNA（NC）,并进行诱导分化培养至第0天和第4 天时,利用荧光定量PCR方法研究分化标志基因过氧化物酶体增殖物激活受体γ（peroxisome proliferator activated receptor γ, PPARγ）、CCAAT增强子结合蛋白（CCAAT/enhancer-binding proteins α, C/EBPα）,脂肪酸代谢关键基因脂肪酸合成酶（fatty acid synthase,FAS）、乙酰辅酶A羧化酶（acetyl-CoA carboxylase α, ACCα）和脂肪酸结合蛋白4（fatty acid binding protein 4, FABP4）基因在牛脂肪细胞分化不同时期干扰KLF3基因处理组和对照组之间表达量的变化,同时在干扰KLF3基因处理并进行诱导脂肪细胞分化的第 4天采用油红O染色法观察干扰KLF3基因处理组和对照组之间脂滴差异及采用酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)方法测定甘油三酯含量进一步确定KLF3基因对牛脂肪细胞分化和脂肪酸代谢的作用。【结果】KLF3-Si2 具有最高的干扰效率达到73%。转染KLF3-Si2后PPARγ的表达量在牛脂肪细胞诱导分化的第0天和第4 天与对照组相比分别下调了58% 和37%,达到了差异极显著（P<0.01）,C/EBPα 的表达量也分别下调了64% 和41%,达到了差异极显著（P<0.01）。脂质代谢的关键基因FABP4的表达量与对照组相比在牛脂肪细胞诱导分化的第0天和第4 天分别下调了89% 和60%,而ACCα的表达量干扰KLF3处理组与对照组相比在脂肪细胞诱导分化第0天和第4天分别下调了50% 和37%, 而FAS的表达量则分别下调了73% 和19%,都分别达到了差异极显著（P<0.01）。在牛脂肪细胞诱导分化第4天油红O染色和甘油三酯含量测定也发现干扰KLF3基因处理组与对照组相比脂滴减少,甘油三酯含量显著下降（P<0.05）。【结论】干扰牛KLF3基因可以抑制牛脂肪细胞分化和脂肪酸代谢关键基因表达量下调,进而影响脂肪的沉积。

关键词: 秦川牛, 前脂肪细胞, KLF3, 脂肪沉积

Abstract:

【Objective】This study aimed to investigate the effects of bovine Krüppel-like factor 3 (KLF3) gene on bovine adipocytes differentiation and fatty acid metabolism, and to explore the effect of KLF3 gene on lipid deposition. 【Method】This research synthesized interference RNA (siRNA) of KLF3, and then transfected KLF3 gene SiRNA and negative to the Qinchuan cattle preadipocytes when the cells grew to a confluence of 70%-90%. The QPCR method was used to determine the expression of adipocyte differentiation marker genes PPARγ and C/EBPα as well the key genes FAS, ACCα and FABP4 in fatty acid metabolism at adipocytes induced differentiation 0 and 4 days when the expression levels of KLF3 gene interfered. At the same time, on the 4th day of inducing adipocyte differentiation after interfering with KLF3 gene treatment, oil red O staining was used to observe the difference of lipid droplets between the KLF3 gene treatment group and the control group, and the enzyme-linked immunosorbent assay (ELISA) method was used to determine the triglyceride content to further determine the effect of the KLF3 gene on bovine adipocyte differentiation and lipid metabolism. 【Result】The results indicated that KLF3-Si2 had the highest interference efficiency, which was 73%. After transfected with KLF3-Si2, on the 0th and 4th day of bovine adipocytes induced differentiation, the expression of PPARγ was extremely significant (P<0.01) down-regulated by 58% and 37%, respectively; the expression of C/EBPα was also extremely significant (P<0.01) down-regulated by 64% and 41%, respectively; the expression level of FABP4, a key gene for lipid metabolism, was down-regulated by 89% and 60%, respectively; while the expression of ACCα interfered with the KLF3 treatment group and the control group was down-regulated by 50% and 37%, respectively; the expression of FAS was down-regulated by 73% and 19%, respectively, and all the difference was extremely significant (P<0.01). On the 4th day of induced differentiation, oil red O staining and triglyceride content determination also showed that the lipid droplets were decreased and the triglyceride content was significantly decreased in the silence KLF3 gene treated group compared with the control group (P<0.05). 【Conclusion】 Interfering with bovine KLF3 gene could inhibit bovine adipocytes differentiation and fatty acid synthesis key genes expression, thus affecting fat deposition.

Key words: Qinchuan cattle, preadipocytes, KLF3, fat deposition

郭红芳,宁越,成功,昝林森. Krüppel-Like Factor 3（KLF3）基因对牛脂肪沉积的作用[J]. 中国农业科学, 2019, 52(7): 1272-1281.

GUO HongFang,NING Yue,CHENG Gong,ZAN LinSen. The Effect of Krüppel-Like Factor 3 (KLF3) Gene on Bovine Fat Deposition[J]. Scientia Agricultura Sinica, 2019, 52(7): 1272-1281.

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引物Primer	引物序列（5′-3′）Sequence（5′-3′）	片段大小Fragment size (bp)	Tm（℃）
KLF3-Si2-F	GCAAAGGAAGCGGAGAAUATT	—	—
KLF3-Si2-R	UAUUCUCCGCUUCCUUUGCTT	—	—
KLF3-Si3-F	GGAAACACACUGGAAUCAATT	—	—
KLF3-Si3-R	UUGAUUCCAGUGUGUUUCCTT	—	—
PPARγ-F	GAGATCACAGAGTACGCCAAG	216	60
PPARγ-R	GGGCTCCATAAAGTCACCAA	216	60
CEBPα-F	ATCTGCGAACACGAGACG	73	60
CEBPα-R	CCAGGAACTCGTCGTTGAA	73	60
ACCα-F	CTCCAACCTCAACCACTACGG	171	60
ACCα-R	GGGGAATCACAGAAGCAGCC	171	60
FAS-F	TAAGGTTCAAATTGCTGCGT	138	60
FAS-R	TCCAGAGCGAAGGAGAGATT	138	60
FABP4-F	TGAGATTTCCTTCAAATTGGG	101	60
FABP4-R	CTTGTACCAGAGCACCTTCATC	101	60
GAPDH-F	CCAACGTGTCTGTTGTGGAT	80	60
GAPDH-R	CTGCTTCACCACCTTCTTGA	80	60
KLF3-F	CAGTCCCTGTCAAGCAAGAG	111	60
KLF3-R	ACAATGGCGTGGAGTAGATG	111	60

物种 Species	序列号 GenBank accession	相似度 Similarity
水牛Bubalusbubalis	XP 006055965.1	99%
绵羊Ovisaries	XP_014962453.1	99%
野猪 Sus scrofa	NP_001127824.1	98%
野骆驼Camelus ferus	XP_006185663.1	98%
智人 Homo sapiens	NP_05057615.3	97%
小家鼠Mus musculus	NP_032479.1	94%