Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (7): 1272-1281.doi: 10.3864/j.issn.0578-1752.2019.07.014

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

The Effect of Krüppel-Like Factor 3 (KLF3) Gene on Bovine Fat Deposition

GUO HongFang1,NING Yue1,CHENG Gong1,2,ZAN LinSen1,2()   

  1. 1 College of Animal Science and Technology, Northwest Agriculture and Forestry University, Yangling 712100, Shaanxi
    2 National Beef Cattle Improvement Center, Yangling, 712100, Shaanxi
  • Received:2018-10-10 Accepted:2019-01-22 Online:2019-04-01 Published:2019-04-04
  • Contact: LinSen ZAN E-mail:zanlinsen@163.com

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

Table 1

Primer used in the study"

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

Table 2

Comparison of bovine KLF3 protein with other GenBank recorded animals"

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%

Fig. 1

Phylogenetic tree of bovine KLF3"

Fig. 2

KLF3 gene expression pattern in Qinchuan newborn cattle tissues (A) and bovine adipocyte(B)"

Fig. 3

KLF3 siRNA screening A. Protein expression when treat with Si2-KLF3; B. KLF3 expression when treat with different SiRNA"

Fig. 4

KLF3 relative expression after interfering with bovine KLF3 gene during adipocyte differentiation ** represented P<0.01. The same as below"

Fig. 5

The relative expression of PPARγ and C/EBPα when silence bovine KLF3 gene during bovine adipocyte differentiation"

Fig. 6

The relative expression of ACCα, FAS and FABP4 when silence bovine KLF3 gene during bovine adipocyte differentiation"

Fig. 7

Oil O staining when silence KLF3 gene at bovine adipocyte induced differentiation 4 days"

Fig. 8

The content of triglyceride in bovine adipocyte when silence KLF3 gene at induced differentiation 4 days"

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