<i>UCP3</i>对民猪前脂肪细胞产热相关基因表达的影响

doi:10.7668/hbnxb.20194780

摘要/Abstract

摘要：

为了探究解偶联蛋白3(UCP3)在民猪不同组织中的表达情况以及对民猪前脂肪细胞中线粒体相关基因和ATP合成酶相关基因表达的影响,采集1月龄民猪的背部脂肪组织作为研究材料,通过酶消化法分离前脂肪细胞进行体外培养,同时采集腋下脂肪、胸部脂肪、背部脂肪、腹股沟脂肪、肾周脂肪和肌内脂肪构建组织表达谱。通过体外转染UCP3基因的过表达载体或干扰片段的方法,采用实时荧光定量PCR技术检测MCU家族基因(MCU、MICU1、MICU2)、ATP合成酶(ATP5B、ATP5E)和1,4,5-三磷酸肌醇受体1型基因(ITPR1)的表达情况以及不同脂肪组织中UCP3基因的表达情况。结果显示:采用酶消化法可以在背部脂肪组织中快速获得足量的前脂肪细胞,细胞边缘折光性良好,边界清晰,形态与成纤维细胞相似。UCP3基因在不同脂肪组织中均有表达,在背部脂肪组织中表达量最高,在腹股沟脂肪中表达量最低。过表达UCP3基因后,MCU家族基因和ITPR1基因表达水平显著升高,ATP5B基因表达水平显著下降。干扰UCP3基因后,MCU家族基因和ITPR1基因表达水平显著降低,ATP合成酶基因表达水平显著升高。结果说明:UCP3基因在不同脂肪组织中存在表达差异,可促进MCU家族基因和ITPR1基因表达、抑制ATP5B基因表达。

关键词: 民猪, 解偶联蛋白3, 实时荧光定量PCR, 前脂肪细胞, 产热相关基因

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

霍秀鹏, 宋照哲, 马红, 汪亮, 刘娣, 郝丽. UCP3对民猪前脂肪细胞产热相关基因表达的影响[J]. 华北农学报, 2024, 39(3): 208-214. doi: 10.7668/hbnxb.20194780.

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.

http://www.hbnxb.net/CN/Y2024/V39/I3/208

图/表 11

表1 UCP3基因CDS区扩增引物

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

表2 干扰引物序列

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

表3 基因定量引物序列

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

图1 民猪前脂肪细胞 A.原代细胞培养24 h;B.原代细胞培养36 h。

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

图2 UCP3在民猪不同脂肪组织表达不同小写字母表示不同组织的差异显著(P<0.05)。

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).

图3 RNA电泳图及UCP3的 CDS区序列PCR扩增 A.RNA电泳图:M.DL2000 Marker;1.RNA。B.UCP3的CDS区序列PCR扩增:M.DL2000 Marker;1.UCP3 CDS区扩增片段。

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.

图4 猪UCP3基因CDS区序列DNAMAN比对结果

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

图5 pcDNA3.1-UCP3质粒双酶切 M1.DL2000 Marker;1.pcDNA3.1-UCP3质粒;2.pcDNA3.1-UCP3质粒双酶切;M2.DL10000 Marker。

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.

图6 siRNA-UCP3干扰引物最佳试验组筛选及重组质粒转染效率检测 A.干扰UCP3基因后细胞形态学观察;B.过表达UCP3后细胞形态学观察;C.最佳干扰浓度筛选;D.UCP3过表达效率检测。不同大写字母表示不同组差异极显著(P<0.01)。

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).

图7 过表达、干扰UCP3后MCU家族基因表达情况 A.过表达UCP3后检测MCU、MICU1、MICU2表达量;B.干扰UCP3后检测MCU、MICU1、MICU2表达量。不同小写字母表示不同组差异显著(P<0.05)。图8同。

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.

图8 过表达、干扰UCP3后ATP5B、ATP5E和ITPR1基因表达情况 A.过表达UCP3后检测ATP5B、ATP5E、ITPR1表达量;B.干扰UCP3后检测ATP5B、ATP5E、ITPR1表达量。

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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UCP3对民猪前脂肪细胞产热相关基因表达的影响

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

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