Effect of UCP3 on the Expression of Heat-related Genes in Min Pig Preadipocytes

doi:10.7668/hbnxb.20194780

Abstract

Abstract:

In order to explore the expression of uncoupling protein 3(UCP3)in different tissues of Min pig and its effect on the expression of mitochondrial-related genes and ATP synthase-related genes in Min pig preadipocytes,the back adipose tissue of 1-month-old Min pig was collected as the research material.The preadipocytes were isolated by enzymatic digestion and cultured in vitro.At the same time,axillary fat,chest fat,back fat,inguinal fat,perirenal fat and intramuscular fat were collected to construct tissue expression profiles.Real-time Quantitative PCR was used to detect the expression of MCU family genes(MCU,MICU1,MICU2),ATP synthase(ATP5B,ATP5E)and 1,4,5-trisphosphate inositol receptor type 1(ITPR1)genes and the expression of UCP3 gene in different adipose tissues by in vitro transfection of UCP3 gene overexpression vector or interference fragment.The results showed that the enzymatic digestion method could quickly obtain sufficient preadipocytes in the back adipose tissue.The cell edge had good refractive index,clear boundary,and similar morphology to fibroblasts.UCP3 gene was expressed in different adipose tissues,with the highest expression in dorsal adipose tissue and the lowest expression in abdominal fat.After overexpression of UCP3 gene,the expression levels of MCU family genes and ITPR1 genes were significantly increased,and the expression level of ATP5B gene was significantly decreased.After UCP3 gene interference,the expression levels of MCU family genes and ITPR1 genes were significantly decreased,and the expression levels of ATP synthase genes were significantly increased.The results showed that UCP3 gene was differentially expressed in different adipose tissues,which could promote the expression of MCU family genes and ITPR1 genes and inhibit the expression of ATP5B gene.

Key words: Min pig, Uncoupling protein 3, Real-time PCR, Preadipocytes, Heat-related genes

HUO Xiupeng, SONG Zhaozhe, MA Hong, WANG Liang, LIU Di, HAO Li. Effect of UCP3 on the Expression of Heat-related Genes in Min Pig Preadipocytes[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(3): 208-214. doi: 10.7668/hbnxb.20194780.

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Figures/Tables 11

Tab.1 Primers for UCP3 gene identification of CDS

引物序列(5'—3') Primer sequence(5'—3')	预扩增片段大小/bp Pre-amplify fragment size	退火温度/℃ Annealing temperature	酶切位点 Restriction sites
F-AAGCTTATGGTGGGCCTGAAGCCT	927	60	Hind Ⅲ
R-GGATCCCAAAACGGAGATTCCCGC			BamH Ⅰ

Tab.2 Interference primer sequence

基因 Gene	引物序列(5'—3') Primer sequence(5'—3')	片段大小/bp Fragment size
sscUCP3-289	F-GACUCCGUCAAGCAGCUUU	21
	R-AAAGCUGCUUGACGGAGUC
sscUCP3-518	F-GCCUGUGGAAAGGAAUUCU	21
	R-AGAAUUCCUUUCCACAGGC
sscUCP3-884	F-GGGCCUUGAUGAAAGUCCA	21
	R-UGGACUUUCAUCAAGGCCC
阴性对照	F-UUCUCCGAACGUGUCACGU	21
Negative control	R-ACGUGACACGUUCGGAGAA

Tab.3 qRT-PCR primer sequence of genes

基因 Gene	引物序列(5'—3') Primer sequence(5'—3')	预扩增片段大小/bp Pre-amplify fragment size	退火温度/℃ Annealing temperature
UCP3	F-CTTCGCCTCCATCCGC	222	60
	R-GCATCCATCGTCCCGCT
MCU	F-ATTTGGGAGTCGTTTATTG	143	60
	R-AGAGTCTGAGATGGGTTTGA
MICU1	F-GAACTTGGTGACAAAGGGAA	305	60
	R-CTCTGAGGAGGTTCTGGTTG
MICU2	F-TGGTGATAAAGGGCTGATT	218	60
	R-CTGTTGGTTCCTGGTGCT
ATP5B	F-ATGGTGGTTACTCCGTGTT	291	60
	R-TGCCCAATAAAGCAGACA
ATP5E	F-GCTACATCCGATACTCCCA	84	60
	R-AAGTCTTCTCGGCATTCG
ITPR1	F-TGCCACGAGTTCCAAAG	118	60
	R-GTAATGCCCTGTAGCAAGAT
GAPDH	F-CGTCCCTGAGACACGATGGT	226	60
	R-CGTCCCTGAGACACGATGGT

Fig.1 Min pig preadipocytes A.Primary cell culture for 24 h; B.Primary cell culture for 36 h.

Fig.2 The expression of UCP3 in different adipose tissues of Min pig Different lowercase letters indicated significant differences in different tissues(P<0.05).

Fig.3 RNA electrophoresis and PCR amplification of UCP3 CDS region sequence A.RNA electrophoresis:M.DL2000 Marker;1.RNA.B.The CDS sequence of UCP3 was amplified by PCR:M.DL2000 Marker;1.Amplified fragment of UCP3 CDS region.

Fig.4 DNAMAN alignment results of porcine UCP3 gene CDS region sequence

Fig.5 pcDNA3.1-UCP3 plasmid double enzyme digestion M1.DL2000 Marker;1.pcDNA3.1-UCP3 plasmid;2.pcDNA3.1-UCP3 plasmid double digestion;M2.DL10000 Marker.

Fig.6 Selection of optimal siRNA-UCP3 interference primers for experimental groups and detection of transfection efficiency of recombinant plasmids A.Morphological observation of cells after interference with UCP3 gene; B.Morphological observation of cells after overexpression of UCP3;C.Screening of optimal interference concentration;D.UCP3 overexpression efficiency detection.Different capital letters indicate that the difference between different groups is extremely significant(P<0.01).

Fig.7 MCU family gene expression after overexpression of interference of UCP3 A.The expression of MCU,MICU1 and MICU2 was detected after overexpression of UCP3;B.The expression levels of MCU,MICU1 and MICU2 were detected after interfering with UCP3.Different lowercase letters indicate significant differences between different groups(P<0.05).The same as Fig.8.

Fig.8 ATP5B,ATP5E and ITPR1 genes expression after overexpression,interference of UCP3 A.The expression levels of ATP5B,ATP5E and ITPR1 were detected after overexpression of UCP3;B.The expression levels of ATP5B,ATP5E and ITPR1 were detected after interfering with UCP3.

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