Cloning,Molecular Characterization and Tissue Expression Analysis of PFKM Gene in Tibetan Sheep
The aim of this study was to analyze the sequence features,bioinformatics and expression patterns of PFKM in muscle tissues of Tibetan sheep at different developmental stages(1 day,3 months,1 year and 3 years).Using the cDNA of longissimus dorsi muscle of adult(3 years old)Tibetan sheep as a template,the coding sequence(CDS)of the PFKM gene was cloned and bioinformatics analysis was performed.Real-time fluorescence quantification(RT-qPCR)was used to detect the expression of the PFKM gene in longissimus dorsi muscle,cardiac muscle,supraspinatus muscle,biceps brachii,lung,liver,spleen and kidney,and PFKM mRNA expression levels were detected in the above tissues at different developmental stages.Western Blot was used to determine the expression level of PFKM protein in the longissimus dorsi muscle at different stages.The results showed that the CDS region of the PFKM gene in Tibetan sheep comprises 2 556 bp and encodes a total of 851 amino acids.Compared with the reference sequence of sheep,there were 19 mutation sites,and it had a close genetic relationship with sheep(98.82%)and goats(98.59%).Predicting result showed that PFKM protein was a hydrophilic protein without signal peptide present,mainly distribute in the cyto-plasm,and the secondary structure of the protein was mainly comprosed of α-helix.There were potential interactions between PFKM protein and 10 glycolytic related protein molecules.The RT-qPCR results showed that with increasing age,the expression of PFKM mRNA in various tissues of Tibetan sheep first increased and then decreased(1 day old<3 months old<1 year old>3 years old),and the expression of PFKM protein in longissimus dorsi muscle was first up-regulated and then down-regulated.This study obtained the complete CDS region of the PFKM gene in Tibetan sheep,and found that it was highly conservated among species and played an important role in muscle growth and develop-ment of Tibetan sheep.
万方数据
维普
