中国农业科学 ›› 2019, Vol. 52 ›› Issue (7): 1272-1281.doi: 10.3864/j.issn.0578-1752.2019.07.014
收稿日期:
2018-10-10
接受日期:
2019-01-22
出版日期:
2019-04-01
发布日期:
2019-04-04
通讯作者:
昝林森
作者简介:
郭红芳,E-mail: 基金资助:
GUO HongFang1,NING Yue1,CHENG Gong1,2,ZAN LinSen1,2()
Received:
2018-10-10
Accepted:
2019-01-22
Online:
2019-04-01
Published:
2019-04-04
Contact:
LinSen ZAN
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。
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.
表1
试验中所用引物"
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 | ||
CEBPα-F | ATCTGCGAACACGAGACG | 73 | 60 |
CEBPα-R | CCAGGAACTCGTCGTTGAA | ||
ACCα-F | CTCCAACCTCAACCACTACGG | 171 | 60 |
ACCα-R | GGGGAATCACAGAAGCAGCC | ||
FAS-F | TAAGGTTCAAATTGCTGCGT | 138 | 60 |
FAS-R | TCCAGAGCGAAGGAGAGATT | ||
FABP4-F | TGAGATTTCCTTCAAATTGGG | 101 | 60 |
FABP4-R | CTTGTACCAGAGCACCTTCATC | ||
GAPDH-F | CCAACGTGTCTGTTGTGGAT | 80 | 60 |
GAPDH-R | CTGCTTCACCACCTTCTTGA | ||
KLF3-F | CAGTCCCTGTCAAGCAAGAG | 111 | 60 |
KLF3-R | ACAATGGCGTGGAGTAGATG |
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