山羊 <i>MZB1</i> 促进肌内脂肪细胞分化

doi:10.7668/hbnxb.20195540

摘要/Abstract

摘要： 为明确山羊边缘区B细胞和B1细胞特异性蛋白(MZB1)在山羊的肌内脂肪分化中起到的调控作用,首先克隆山羊 MZB1 基因实际序列,并对序列进行生物信息学分析获得相关生物学特征;随后利用实时荧光定量PCR(qPCR)绘制山羊 MZB1 组织与细胞时序表达谱;最后转染山羊MZB1过表达质粒至细胞内并进行诱导分化,通过形态学观察及 qPCR 等试验确定过表达山羊MZB1基因对该分化过程的影响。结果表明:山羊MZB1基因序列全长共计860 bp,CDS 区长度为570 bp;山羊 MZB1 基因共编码189 aa;山羊MZB1蛋白属于酸性蛋白且带负电,该蛋白在细胞中可以稳定存在。山羊心脏、肝脏等8个组织均检测到山羊MZB1表达且在脾脏中表达量最高。在油酸诱导山羊肌内脂肪细胞分化72 h后,山羊MZB1表达量极显著高于诱导分化前。山羊 MZB1过表达增加山羊肌内脂肪细胞中的脂滴数量和大小;FASN、PPARγ、C/EBPα、SREBP1、C/EBPβ表达水平极显著上调。以上结果表明,MZB1促进山羊肌内脂肪细胞分化,且这种促进作用可能通过上调脂肪分化标志基因PPARγ、C/EBPα、C/EBPβ,脂肪酸合成标志基因SREBP1、FASN的表达来实现。

关键词: 山羊, MZB1, 肌内脂肪细胞, 过表达, 细胞分化

Abstract:

In order to clarify the regulatory role of marginal zone B and B1 cell specific protein(MZB1)in intramuscular adipose differentiation of goats,the study first cloned the full-lenth sequence of goat MZB1 gene and analyzed the sequence to obtain the relevant biological characteristics;then used Real-time Quantitative PCR(qPCR)to construct the expression profile of goat MZB1 in tissues and cells;finally,the goat MZB1 overexpression plasmid was transfected into the target cells,followed by differentiation induction;the effects of MZB1 overexpression on the differentiation process were evaluated through morphological analysis and qPCR.The results showed that the goat MZB1 gene sequence was 860 bp in length,including 570 bp CDS region,encoding 189 amino acids;goat MZB1 protein was not only acidic but also negatively charged,which could be stably existed in the cells.Goat MZB1 expression was detected in eight tissues,including the heart and the liver,and the highest expression was found in the spleen.The expression of MZB1 in goat intramuscular adipocytes after 72 h of oleic acid-induced differentiation was significantly higher than that before oleic acid-induced differentiation.Goat MZB1 overexpression increased the number and size of lipid droplets in goat intramuscular adipocytes;the expression levels of FASN,PPARγ,C/EBPα,SREBP1,and C/EBPβ were highly significantly upregulated.The above results suggest that MZB1 plays a positive regulatory role in goat intramuscular adipocyte differentiation,and this positive regulation may be achieved by increasing the expression of the adipose differentiation marker genes PPARγ,C/EBPα,C/EBPβ,and the fatty acid synthesis marker genes SREBP1 and FASN.

Key words: Goat, MZB1, Intramuscular fat cells, Overexpression, Cell differentiation

中图分类号:

S827

董耀徽, 王永, 李艳艳, 王金玲, 王友利, 刘伟, 邢佳妮, 林亚秋. 山羊 MZB1 促进肌内脂肪细胞分化[J]. 华北农学报, 2025, 40(5): 216-223. doi: 10.7668/hbnxb.20195540.

DONG Yaohui, WANG Yong, LI Yanyan, WANG Jinling, WANG Youli, LIU Wei, XING Jiani, LIN Yaqiu. Goat MZB1 Promotes Intramuscular Adipocyte Differentiation[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(5): 216-223. doi: 10.7668/hbnxb.20195540.

http://www.hbnxb.net/CN/Y2025/V40/I5/216

图/表 9

表1 相关引物信息

Tab.1 Relevant primer information

基因 Gene	引物序列(5'—3') Primer sequence(5'—3')	Tm/℃	产物长度/bp Product length	用途 Application
MZB1	S:CAGAGAGGAACCCACACACC	60	707	PCR
	A:CAGAAAGGAGGGAGGCAGAT
MZB1	S:CTATGGAGTTGGAGAAGTGGAC	58	261	qPCR
	A:TCTGTCCTTGTGTCTGGAACCT
UXT	S: ATGGAGTCCTTGGTGAGGTTGT	60	182	内参
	A:GCAAGTGGATTTGGGCTGTAAC
C/EBPα	S: AGGCGGTCATTGTCACTGGT	58	142	qPCR
	A: CCGTGGACAAGAACAGCAAC
PPARγ	S:AAGCGTCAGGGTTCCACTATG	60	197	qPCR
	A:GAACCTGATGGCGTTATGAGAC
C/EBPβ	S:CAAGAAGACGGTGGACAAGC	66	204	qPCR
	A:AACAAGTTCCGCAGGGTG
LPL	S:TCCTGGAGTGACGGAATCTGT	60	174	qPCR
	A:GACAGCCAGTCCACCACGAT
SREBP1	S:AGTGGTGGGCCTCTCTGA	58	127	qPCR
	A:GCAGGGGTTTCTCGGACT

表2 生物信息学分析软件

Tab.2 Software for bioinformatics analysis

分析内容 Analysis content	工具(网址) Tool(Website)
寻找开放阅读框(ORF)Finding open reading frame (ORF)	ORF finder(https://www.bioinformatics.org/sms2/orf_find.html)
获取氨基酸序列Amino acid sequence acquisition	(https://www.ncbi.nlm.nih.gov/orffinder/)
信号肽位点预测Signal peptide site prediction	SignalP-5.0 Server(https://services.healthtech.dtu.dk/services/SignalP-5.0/)
蛋白质理化性质分析	Protparam(https://web.expasy.org/protparam/)
Analysis of physical and chemical properties of proteins
跨膜结构域预测Transmembrane domain prediction	TMHMM 2.0(https://services.healthtech.dtu.dk/services/TMHMM-2.0/)
功能结构域预测Functional domain prediction	Conserved Domain(https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi)
磷酸化、糖基化位点预测	Netphos、NetOGlyc、NetNGlyc(https://services.healthtech.dtu.dk/services/NetPhos-3.1/、
Prediction of phosphorylation and glycosylation sites	https://services.healthtech.dtu.dk/services/NetNGlyc-1.0/、 https://services.
	healthtech.dtu.dk/services/NetPhos-3.1/)
蛋白质二级结构预测Protein secondary structure prediction	SOPMA(https://npsa.lyon.inserm.fr/cgibin/npsa_automat.pl?page=/NPSA/npsa_sopma.html)
相互作用蛋白质预测Interaction protein prediction	STRING数据库(https://cn.string-db.org/)
蛋白质三级结构预测Tertiary structure prediction of proteins	SWISS-MODEL(https://swissmodel.expasy.org/)

图1 山羊MZB1基因克隆 A.山羊 MZB1 基因克隆:M.DL2000 Marker,G.MZB1基因;B.山羊MZB1基因编码的氨基酸序列;C.氨基酸序列相似性比对图。

Fig.1 Goat MZB1 gene clone A.Goat MZB1 gene cloning: M.DL2000 Marker,G.MZB1 gene; B.Amino acid sequences encoded by the goat MZB1 gene; C.Amino acid sequence similarity comparison plot.

表3 组成山羊MZB1蛋白的氨基酸

Tab.3 Amino acids composing goat MZB1 protein

氨基酸 Amino acid	数目/个 Amount	百分比/% Percentage	氨基酸 Amino acid	数目/个 Amount	百分比/% Percentage
Ala	14	7.4	Leu	27	14.3
Arg	10	5.3	Lys	6	3.2
Asn	0	0.0	Met	7	3.7
Asp	10	5.3	Phe	3	1.6
Cys	6	3.2	Pro	11	5.8
Gln	10	1.7	Ser	12	6.3
Glu	15	7.9	Thr	12	6.3
Gly	20	10.6	Trp	4	2.1
His	5	2.6	Tyr	5	2.6
Ile	4	2.1	Val	8	4.2
Pyl	0	0.0	Sec	0	0.0

图2 山羊MZB1蛋白性质分析 A.MZB1信号肽预测;B.MZB1蛋白修饰位点;C.MZB1跨膜结构域预测;D.MZB1亲疏水性验证。

Fig.2 Characteristics analysis of goat MZB1 protein A.Prediction of MZB1 signal peptide; B.MZB1 protein modification sites; C.Prediction of goat MZB1 transmembrane structural domains; D.Verification of goat MZB1 hydrophilicity.

图3 山羊MZB1蛋白结构分析 A.MZB1二级结构;B.MZB1三级结构;C.山羊MZB1互作蛋白;D.山羊MZB1亚细胞定位。

Fig.3 Goat MZB1 protein structural analysis A.MZB1 secondary structure forecasts;B.MZB1 tertiary structure forecasts;C.MZB1 interaction protein prediction; D.Subcellular localization of goat MZB1 protein.

图4 山羊MZB1在8个山羊组织部位和油酸诱导成脂分化不同时间肌内脂肪细胞中的表达量不同大写字母表示差异极显著(P<0.01)。图5—6同。

Fig.4 Expression of goat MZB1 in eight goat tissue sites and intramuscular adipocytes at different times of oleic acid-induced lipogenic differentiation Differences with different uppercase letters indicate highly significant differences(P<0.01).The same as Fig.5—6.

图5 山羊 MZB1过表达效率及形态学观察 A.山羊 MZB1基因过表达效率;B.形态学观察(200×)。

Fig.5 Overexpression efficiency and morphologic observation of goat MZB1 A.Overexpression efficiency of goat MZB1 gene;B.Morphological observation(200×).

图6 山羊脂肪细胞分化标志基因相对表达量分析 A.过表达 MZB1 对脂肪细胞分化标志基因及甘油三酯合成标志基因的影响;B.过表达 MZB1对甘油三酯分解标志基因及甘油三酯合成标志基因的影响。

Fig.6 The expression level of genes related to adipocyte differentiation A.Overexpression MZB1 affects adipocyte differentiation marker gene and triglyceride synthesis marker gene; B.Overexpression of MZB1 affects triglyceride catabolism marker gene and triglyceride synthesis marker gene.

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山羊 MZB1 促进肌内脂肪细胞分化

Goat MZB1 Promotes Intramuscular Adipocyte Differentiation

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