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

doi:10.7668/hbnxb.20195346

Abstract

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

CLC Number:

S823.85

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.

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

Figures/Tables 9

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

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

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.

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.

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.

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

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

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.

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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