Cloning and expression profile analysis of CDS sequence of m⁶A methyltransferase METTL14 gene from Hu sheep

【Objective】Coding region (CDS) sequence of methyltransferase-like protein 14 gene (METTL14) in Hu sheep was cloned,which helped to explore its molecular characteristics and tissue expression distribution,and lay a foun-dation for revealing the specific molecular mechanism of METTL14 gene regulating muscle development in Hu sheep.【Method】RNA was extracted from the muscle tissue of Hu sheep and cDNA was synthesized by reverse transcription.The CDS sequence of METTL14 gene was amplified by using cDNA as template.Bioinformatics analysis was carried out by online softwares,such as ProtParam,ProtScale,and SignaIP-6.0.The tissue expression profile of METTL14 gene in Hu sheep was analyzed by real-time fluorescence quantitative PCR.【Result】The total length of the CDS region of Hu sheep METTL14 gene was 1371 bp,which encoded 456 amino acids residues.The CDS sequence of METTL14 gene in Hu sheep was 89.28%-99.78%similar to that in 12 reference species,including Bos taurus,Homo sapiens,Mus musculus,Rattus norvegicus,Macaca nemestrina,Capra hircus,Oryx dammah,Equus caballus,Camelus bactrianus,Bos mutus,Sus scrofa,Bubalus bubalis,and the corresponding amino acid sequence similarity of METTL14 was 98.66%-100.00%.The phylogenetic tree constructed based on the nucleotide sequence similarity of METTL14 gene showed that Hu sheep had the closest relationship with goats and far relationship with bactrian camels.The molecular formula of Hu sheep METTL14 protein was C₂₂₇₇H₃₅₇₅N₆₆₉O₇₀₉S₁₆,the molecular weight was 52179.45 Da,and the theoretical isoelectric point(pI) was 5.89.There was no signal peptide and no transmembrane domain.It had strong hydrophilicity and was an un-stable protein,mainly distributed in the nucleus (69.6%).The METTL14 protein contained 12 α-helices and 10 β-folds,but it was mainly composed of random coils.The METTL14 gene was expressed in the heart,liver,spleen,lung,kid-ney,brain,testis,muscle,and subcutaneous fat of Hu sheep,with the highest relative expression in the heart and the lowest relative expression in the liver.【Conclusion】As the core protein of m⁶ A methylation,METTL14 is highly con-served during species evolution.The METTL14 gene is expressed in several tissues including muscle and fat of Hu sheep,and the expression level is related to the metabolic activity of the organism,which can be investigated as a candidate gene to regulate the muscle growth and development of Hu sheep.