牦牛<i>MAP3K7</i>基因克隆及其与皮下前体脂肪细胞分化关系分析

doi:10.7668/hbnxb.20195346

摘要/Abstract

摘要：

MAP3K7是MAP3K家族中的一员,在脂肪分化、炎症和肿瘤发生过程中发挥着重要作用,但其与牦牛脂肪分化的关系未见报道,并且其基因序列未知,制约了相关功能研究。为了获得牦牛MAP3K7 基因编码区 (CDS) 保守区域序列,明确其表达特征,了解其在牦牛不同组织中的表达水平及其与牦牛皮下前体脂肪细胞分化的相关性,利用RT-PCR技术,克隆牦牛 MAP3K7 基因并利用生物信息学方法分析生物学特性;利用实时荧光定量 PCR (qPCR) 技术检测MAP3K7 在牦牛心、肝、脾等10个组织中的表达情况,与不同分化阶段牦牛皮下脂肪细胞中MAP3K7基因和脂分化相关基因的表达情况,同时结合油红O染色,明确MAP3K7 基因对牦牛皮下前体脂肪细胞分化的影响。结果表明:克隆所得牦牛MAP3K7基因CDS区长度为1 740 bp,编码579个氨基酸,与野牦牛MAP3K7 预测序列CDS区比对发现第967位 A>G,导致第323位氨基酸Thr>Ala的突变。理化性质分析发现,该蛋白为整体带负电的亲水酸性蛋白,具有112个氨基酸磷酸化位点,其氨基酸组成中,占比最高的是丝氨酸;二级结构预测显示,其无规卷曲含量最高,可能与TAB3等蛋白质间存在相互作用关系。核苷酸同源性比对与进化树结果表明,牦牛MAP3K7在反刍动物间具有较高的保守性,与野牦牛同源性最高且进化关系最近。组织定量检测发现,MAP3K7在牦牛皮下脂肪和内脏脂肪中的表达量相对较低,在牦牛皮下前体脂肪细胞诱导分化过程中,MAP3K7表达量随着成熟脂肪细胞的增多先上调,至分化到一定程度后下调。综上,MAP3K7基因在牦牛皮下前体脂肪细胞分化前期高表达以促进分化,但当分化到一定程度时,表达下调抑制进一步分化。

关键词: 牦牛, MAP3K7, 基因表达, 皮下脂肪, 脂肪分化

Abstract:

MAP3K7 is a member of the MAP3K family and plays an important role in fat differentiation,inflammation and tumorigenesis.However,its relationship with yak fat differentiation has not been reported,and its gene sequence is unknown,which restricts the related functional research.In order to obtain the conserved region sequence of yak MAP3K7 gene coding region (CDS),clarify its expression characteristics,and understand its expression level in different tissues of yak and its correlation with subcutaneous preadipocyte differentiation of yak,the MAP3K7 gene of yak was cloned by RT-PCR and the biological characteristics were analyzed by bioinformatics methods.Real-time Fluorescence Quantitative PCR (qPCR) was used to detect the expression of MAP3K7 in 10 tissues of yak heart,liver,spleen,etc,and the expression of MAP3K7 gene and lipid differentiation-related genes in yak subcutaneous adipocytes at different differentiation stages.At the same time,combined with oil red O staining,the effect of MAP3K7 gene on the differentiation of yak subcutaneous preadipocytes was clarified.The results showed that the length of the CDS region of the cloned yak MAP3K7 gene was 1 740 bp,encoding 579 amino acids.Compared with the CDS region of the wild yak MAP3K7 predicted sequence,it was found that A>G at position 967,resulting in a mutation of Thr>Ala at position 323.The analysis of physical and chemical properties showed that the protein was a negatively charged hydrophilic acidic protein with 112 amino acid phosphorylation sites.Among its amino acid composition,serine accounted for the highest proportion.The secondary structure prediction showed that the content of random coil was the highest,which might interact with proteins such as TAB3.The results of nucleotide homology comparison and phylogenetic tree showed that yak MAP3K7 was highly conserved among ruminants,and had the highest homology and the closest evolutionary relationship with wild yak.Tissue quantitative detection showed that the expression of MAP3K7 in subcutaneous fat and visceral fat of yak was relatively low.During the differentiation of subcutaneous preadipocytes of yak,the expression of MAP3K7 was up-regulated with the increase of mature adipocytes,and then down-regulated after differentiation to a certain extent.In summary,MAP3K7 gene was highly expressed in the early stage of differentiation of yak subcutaneous preadipocytes to promote differentiation,but when the differentiation reached a certain level,the down-regulation of expression inhibited further differentiation.

Key words: Bos grunniens, MAP3K7, Gene expression, Subcutaneous fat, Adipose differentiation

中图分类号:

S823.85

蓝湘一, 何洋洋, 张明, 黄慈, 王海波, 王会, 柴志欣, 钟金城. 牦牛MAP3K7基因克隆及其与皮下前体脂肪细胞分化关系分析[J]. 华北农学报, 2025, 40(3): 225-233. doi: 10.7668/hbnxb.20195346.

LAN Xiangyi, HE Yangyang, ZHANG Ming, HUANG Ci, WANG Haibo, WANG Hui, CHAI Zhixin, ZHONG Jincheng. Cloning of Yak MAP3K7 Gene and Its Relationship with the Differentiation of Subcutaneous Preadipocytes[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(3): 225-233. doi: 10.7668/hbnxb.20195346.

http://www.hbnxb.net/CN/Y2025/V40/I3/225

图/表 9

表1 引物信息

Tab.1 Primers information

目的基因 Target gene	引物序列 (5'—3') Primer sequence (5'—3')	退火温度/℃ Tm	产物长度/bp Product length	用途 Utilization
MAP3K7	F:TCCAGGTTGAAGGTCGGT	57	1 902	PCR
	R:CACGCCAAAAAGCTAACACT
MAP3K7	F:TTGCCATATTACACCGCTGC	60	120	qPCR
	R:GTTTGGTGGTTTCAGGTCCC
β-actin	F:TGATGATATTGCTGCGCTCG	60	153	qPCR
	R:TACGAGTCCTTCTGGCCCAT
C/EBPα	F:GTGGACAAGAACAGCAACGAGTA	60	138	qPCR
	R: GCGGTCATTGTCACTGGTCAG
PPARγ	F: CCAAATATCGGTGGGAGTCG	60	107	qPCR
	R: ACAGCGAAGGGCACACTCTC
FASN	F: CGAGTCCCCTGACCACTATCTG	60	130	qPCR
	R: AGGCGTGTGCTCCATGTTCT

表2 序列分析工具及相应分析内容

Tab.2 Sequence analysis tools and corresponding analysis content

软件工具Software tools	分析内容Analysis content
ORFfinder(https://www.ncbi.nlm.nih.gov/orffinder/)	寻找基因开放阅读框 (ORF) 获取氨基酸序列
Blast(https://blast.ncbi.nlm.nih.gov/Blast.cgi)	牦牛预测序列及其他物种序列对比
DNAMAN	物种间氨基酸序列比对
MEGA 5.0	系统进化树构建
Protparam(http://web.expasy.org/protparam/)	蛋白质理化性质分析
Netphos(http://www.cbs.dtu.dk/services/NetPhos)	磷酸化位点预测
Conserved Domain (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi)	功能结构域预测
SOPMA(https://npsa-prabi.ibep.fr/cgi-bin/)	蛋白质二级结构预测
SWISS-MODEL(https://swissmodel.expasy.org/)	蛋白质三级结构预测
STRING(https://string-db.org/)	相互作用蛋白质预测

图1 牦牛MAP3K7基因序列分析 A:M.DL2000 DNA Marker,1.MAP3K7 基因;B:上行表示牦牛MAP3K7核苷酸序列,下行表示对应的氨基酸序列,加粗表示丝氨酸磷酸化位点,三角形表示苏氨酸磷酸化位点,圆形表示酪氨酸磷酸化位点,正方形表示丙氨酸磷酸化位点,矩形框着的ATG表示起始密码子,矩形框着的TGA表示终止密码子;C:野牦牛MAP3K7 部分核酸预测序列 (XM _005889275.2) 与本研究获得的牦牛MAP3K7 部分核酸序列(PQ058292)和推导氨基酸比较,矩形框着潜在的多肽位 (G967A) 和氨基酸相应变化。

Fig.1 Sequence analysis of yak MAP3K7 gene A:M.DL2000 DNA Marker,1.MAP3K7 gene;B:The upstream represents the yak MAP3K7 nucleotide sequence,and the downstream represents the corresponding amino acid sequence,thickening represents serine phosphorylation site,the triangle represents the threonine phosphorylation site,the circle indicates the tyrosine acidification site,the square represents the alanine phosphorylation site,the ATG framed by the rectangle represents the start codon,the TGA framed by the rectangle represents the termination codon;C:Partial nucleic acid prediction sequence of wild yak MAP3K7 (XM_005889275.2) compared with the partial nucleic acid sequence(PQ058292)and deduced amino acid of yak MAP3K7 obtained in this study,the rectangular frame contains potential peptide position (G967A) and corresponding amino acid changes.

图2 牦牛MAP3K7蛋白氨基酸组成与疏水性直观图 A.牦牛MAP3K7蛋白疏水性直观图预测,图中 Score>0 疏水性,Score<0 亲水性;B.牦牛MAP3K7蛋白氨基酸组成。

Fig.2 Diagram of amino acid composition and hydrophobicity of yak MAP3K7 protein A.Hydrophobicity intuitive map prediction of MAP3K7 protein in yak,Score>0 hydrophobic,Score<0, hydrophilic;B.Amino acid composition of yak MAP3K7 protein.

图3 牦牛 MAP3K7蛋白结构特征分析 A.牦牛MAP3K7氨基酸序列生物学功能预测;B.MAP3K7氨基酸序列二级结构预测:长度从长到短分别代表 α-螺旋、延伸链、β-折叠及无规则卷曲。

Fig.3 Structural feature analysis of MAP3K7 protein in yak A.Biological function prediction of yak MAP3K7 amino acid sequence;B.The secondary structure prediction of MAP3K7 amino acid sequence: the length from long to short represents alpha helix,extended strand,beta turn and random coil,respectively.

图4 牦牛MAP3K7 蛋白与其他蛋白互作网络

Fig.4 The interaction network between yak MAP3K7 protein and other proteins

图5 牦牛MAP3K7核苷酸序列同源性比较及进化树构建

Fig.5 Homology comparison and phylogenetic tree construction of yak MAP3K7 nucleotide sequence

图6 MAP3K7基因在牦牛不同组织中的相对表达水平不同小写字母表示差异显著 (P<0.05)。图7同。

Fig.6 Relative expression levels of yak MAP3K7 gene in different tissues The difference between different lowercase letters was significant(P<0.05).The same as Fig.7.

图7 MAP3K7基因与牦牛皮下前体脂肪细胞分化的关系 A.PPARγ 基因的分化时序性表达;B.C/EBPα 基因的分化时序性表达;C.FASN基因在牦牛皮下前体脂肪细胞分化过程中的相对表达量;D.MAP3K7基因的分化时序性表达;E.分化阶段油红O染色(100×);F.分化阶段油红O染色OD值 (510 nm)。

Fig.7 The relationship between MAP3K7 gene and the differentiation of yak subcutaneous preadipocytes A.PPARγ gene differentiation timing expression;B.The differentiation timing expression of C/EBPα gene;C.Relative expression of yak FASN gene in the differentiation of subcutan-eous preadipocytes;D.The differentiation timing expression of MAP3K7 gene;E.Oil red O staining at differentiation stage(100×);F.The OD value of oil red O staining at differentiation stage (510 nm).

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牦牛MAP3K7基因克隆及其与皮下前体脂肪细胞分化关系分析

Cloning of Yak MAP3K7 Gene and Its Relationship with the Differentiation of Subcutaneous Preadipocytes

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牦牛MAP3K7基因克隆及其与皮下前体脂肪细胞分化关系分析

Cloning of Yak MAP3K7 Gene and Its Relationship with the Differentiation of Subcutaneous Preadipocytes

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