青海湖裸鲤Myostatin基因克隆及组织表达特性

doi:10.7668/hbnxb.20196557

摘要/Abstract

摘要：

克隆青海湖裸鲤MSTN基因的全长cDNA序列,明确该基因在青海湖裸鲤不同组织中的表达特性,以及在不同鲤科鱼类肌肉组织中的表达差异,为探讨青海湖裸鲤生长缓慢的分子机制提供基础数据。采用5'RACE和3'RACE技术克隆获得青海湖裸鲤MSTN基因全长cDNA序列,通过生物信息学方法分析其序列特征,并利用RT-qPCR技术检测该基因在青海湖裸鲤不同组织及不同鲤科鱼类肌肉组织中的差异性表达。结果表明:青海湖裸鲤MSTN基因cDNA序列全长为2 168 bp,其中开放阅读框长度为1 128 bp,可编码375 个氨基酸,以ATG为起始密码子,TGA为终止密码子,预测编码蛋白含有70 个苏氨酸(Thr)磷酸化位点。系统发育分析显示,青海湖裸鲤MSTN基因与其他鲤科鱼类同源基因聚为一支,亲缘关系较近。qPCR结果表明,MSTN在青海湖裸鲤的鳃、肾脏、肝脏、心脏、脑、肌肉及皮肤组织中均有表达但在脑和肌肉组织中表达量较高。不同鲤科鱼类中,MSTN基因在青海湖裸鲤和花斑裸鲤中的表达量相对较高,在草鱼、鲫鱼、武昌鱼表达量相对较低。成功克隆获得青海湖裸鲤MSTN基因的全长cDNA序列,明确了其序列特征与进化地位,揭示了该基因在青海湖裸鲤不同组织及不同鲤科鱼类间的表达差异。

关键词: 青海湖裸鲤, Myostatin, 基因克隆

Abstract:

To clone the full-length cDNA sequence of the MSTN(Myostatin)gene from Gymnocypris przewalskii,clarify its expression characteristics in different tissues of G.przewalskii and expression differences in the muscle tissues of different Cyprinidae fishes,and provide basic data for exploring the molecular mechanism underlying the slow growth of G.przewalskii.5'RACE and 3'RACE techniques were used to clone the full-length cDNA sequence of the MSTN gene from G.przewalskii.Bioinformatics methods were employed to analyze its sequence characteristics,and Quantitative Real-time PCR(RT-qPCR)was used to detect the differential expression of this gene in different tissues of G.przewalskii and muscle tissues of different Cyprinidae fishes.The results showed that the full-length cDNA sequence of the MSTN gene from G.przewalskii was 2 168 bp,including an open reading frame(ORF)of 1 128 bp encoding 375 amino acids,with ATG as the start codon and TGA as the stop codon.The predicted encoded protein contained 70 threonine(Thr)phosphorylation sites.Phylogenetic analysis showed that the MSTN gene of G.przewalskii clustered with homologous genes of other Cyprinidae fishes,indicating a close genetic relationship.RT-qPCR results revealed that MSTN was expressed in gill,kidney,liver,heart,brain,muscle,and skin tissues of G.przewalskii,with relatively high expression levels in the brain and muscle tissues.Among different Cyprinidae fishes,the expression level of the MSTN gene was relatively higher in G.przewalskii and Gymnocypris eckloni,while relatively lower in Ctenopharyngodon idella,Carassius auratus,and Megalobrama amblycephala.The full-length cDNA sequence of the MSTN gene from G.przewalskii was successfully cloned,and its sequence characteristics and evolutionary status were clarified,the expression differences of this gene in different tissues of G.przewalskii and among different Cyprinidae fishes were revealed.

Key words: Gymnocypris przewalskii, Myostatin, Gene cloning

中图分类号:

李昭楠, 张晶晶, 李长忠, 梁天秀, 黄书晨, 杨颖, 金文杰, 陈艳霞. 青海湖裸鲤Myostatin基因克隆及组织表达特性[J]. 华北农学报, 2026, 41(4): 231-238. doi: 10.7668/hbnxb.20196557.

LI Zhaonan, ZHANG Jingjing, LI Changzhong, LIANG Tianxiu, HUANG Shuchen, YANG Ying, JIN Wenjie, CHEN Yanxia. Cloning and Tissue Expression Characteristics of Myostatin Gene in Gymnocypris przewalskii[J]. Acta Agriculturae Boreali-Sinica, 2026, 41(4): 231-238. doi: 10.7668/hbnxb.20196557.

http://www.hbnxb.net/CN/Y2026/V41/I4/231

图/表 11

表1 PCR引物信息

Tab.1 PCR primer information

引物名称 Name of primers	引物序列(5'—3') Sequence of primers(5'—3')	用途 Usage
3'-RACE-F	TTGCTAAAAATGTTGTAAAAACAGATT	3'RACE基因特异性引物
3'-RACE-R	ATGTTTGCTAATTGTATTGTATACTTGC
5'-RACE-F	ATGTAGTCGCATCAGCTTGCTGT	5'RACE基因特异性引物
5'-RACE-R	CGTGGCTGTGGAAGGCTG
MSTN1-F	CAGCAAACTCCGACTCAA	PCR扩增引物
MSTN1-R	TCGTATGTGCCTTCCTCC
β-actin-F	TTCTTGGGTATGGAGTCTTG	内参引物
β-actin-R	AGTATTTACGCTCAGGTGGG
MSTN-F	CCCCATCGTTCAAGTAGATCGGAA	表达检测引物
MSTN-R	CTATACTCTGCCAAGATGTCACTCC

图1 青海湖裸鲤MSTN基因CDS序列基因克隆 A.PCR扩增产物;B.菌落PCR扩增产物。

Fig.1 Gymnocypris przewalskii MSTN gene sequences of CDS gene clone A.PCR amplification products;B.Colony PCR amplification products.

图2 RACE扩增结果 A.5'-RACE扩增产物;B.3'-RACE扩增产物;C.全长cDNA。

Fig.2 RACE amplification result A.5'-RACE amplification products;B.3'-RACE amplification product;C.MSTN full-length cDNA.

图3 青海湖裸鲤基因CDS区序列

Fig.3 CDS sequences of Gymnocypris przewalskii

表2 MSTN蛋白氨基酸组成及比例

Tab.2 Amino acid composition and proportion of MSTN protein

氨基酸 Amino acids	数量 Numbers	比例/% Percentage	氨基酸 Amino acids	数量 Numbers	比例/% Percentage
Leu	29	7.70	Val	21	5.60
Ser	27	7.20	Arg	20	5.30
Asp	25	6.70	Glu	20	5.30
Ile	25	6.70	Cys	14	3.70
Ala	23	6.10	Asn	12	3.20
Lys	23	6.10	Met	12	3.20
Pro	23	6.10	His	11	2.90
Gln	21	5.60	Phe	11	2.90
Gly	21	5.60	Tyr	10	2.70
Thr	21	5.60	Trp	6	1.60

图4 青海湖裸鲤MSTN蛋白亲疏水性预测

Fig.4 Prediction of the hydrophilicity of MSTN protein in Gymnocypris przewalskii

图5 青海湖裸鲤MSTN蛋白磷酸化位点预测

Fig.5 Prediction of MSTN protein phosphorylation site of Gymnocypris przewalskii

图6 青海湖裸鲤MSTN蛋白结构域预测

Fig.6 Prediction of MSTN protein domain of Gymnocypris przewalskii

图7 青海湖裸鲤MSTN蛋白的二级结构(A)和三级结构(B)预测

Fig.7 Prediction of the secondary structure(A)and tertiary structure(B)of MSTN protein in Gymnocypris przewalskii

图8 基于MSTN氨基酸序列构建系统进化树

Fig.8 Construction of phylogenetic tree based on MSTN amino acid sequence

图9 MSTN基因RT-qPCR检测 A.青海湖裸鲤MSTN基因的组织特异性相对mRNA水平;B.不同鲤科鱼类MSTN基因相对mRNA水平。不同小写字母表示差异显著(P<0.05),相同表示差异不显著(P>0.05)。

Fig.9 RT-qPCR analysis of MSTN gene A.Tissue-specific relative mRNA expression of MSTN in Gymnocypris przewalskii;B.Relative mRNA expression of MSTN in different Cyprinidae fishes. Different lowercase letters indicate significant differences(P<0.05),and the same letters indicate no significant differences(P>0.05).

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