Cloning and Tissue Expression Characteristics of Myostatin Gene in Gymnocypris przewalskii

doi:10.7668/hbnxb.20196557

Abstract

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

CLC Number:

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.

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Figures/Tables 11

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

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

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

Fig.3 CDS sequences of Gymnocypris przewalskii

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

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

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

Fig.6 Prediction of MSTN protein domain of Gymnocypris przewalskii

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

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

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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