藏绵羊PFKM基因克隆、生物信息学及组织表达分析  

Cloning,Molecular Characterization and Tissue Expression Analysis of PFKM Gene in Tibetan Sheep

作　　者：雒瑞瑞 齐兴财 刘子龙 李讨讨 王彩莲[1] 郎侠[1] 宋淑珍[1] LUO Ruirui;QI Xingcai;LIU Zilong;LI Taotao;WANG Cailian;LANG Xia;SONG Shuzhen(Animal Husbandry,Pasture and Green Agriculture Institute,Gansu Academy of Agricultural Sciences,Lanzhou 730070,China;College of Animal Science and Technology,Gansu Agricultural University,Lanzhou 730070,China)

机构地区：[1]甘肃省农业科学院畜草与绿色农业研究所,兰州730070 [2]甘肃农业大学动物科学技术学院,兰州730070

出　　处：《中国草食动物科学》2024年第5期1-10,共10页China Herbivore Science

基　　金：甘肃省科技计划资助项目(23JRRA1338);甘肃省农业科学院科研条件建设及成果转化项目(博士基金)(2022GAAS60)。

摘　　要：本研究旨在分析藏绵羊PFKM的序列特征、生物信息学及其在不同发育阶段(1日龄、3月龄、1岁龄和3岁龄)肌肉组织中的表达模式。以成年(3岁龄)藏绵羊的背最长肌cDNA为模板,克隆PFKM基因的编码区序列,并进行生物信息学分析。利用实时荧光定量(RT-qPCR)技术检测PFKM基因在背最长肌、心肌、冈上肌、肱二头肌、肺脏、肝脏、脾脏和肾脏中的表达情况,并检测不同发育阶段上述组织中PFKM mRNA表达水平的变化。同时,用Western Blot检测不同年龄阶段藏绵羊背最长肌中PFKM蛋白的表达水平。结果表明,藏绵羊PFKM基因的CDS区全长2 556 bp,共编码851个氨基酸,与绵羊参考序列相比存在19个突变位点,与绵羊(98.82%)和山羊(98.59%)具有较近的亲缘关系;预测PFKM蛋白为亲水性蛋白,不存在信号肽,主要分布在细胞质中,蛋白的二级结构以α-螺旋为主,PFKM蛋白与10个糖酵解相关蛋白质分子间存在潜在的相互作用;RT-qPCR结果显示,随着年龄的增加,PFKM mRNA在藏绵羊各组织中基本呈现先上升后下降(1日龄<3月龄<1岁龄>3岁龄)的表达趋势,背最长肌中PFKM蛋白的表达也先上调后下调。本研究获得了藏绵羊PFKM基因完整的CDS区,发现其在物种间的保守性较高,并在藏绵羊肌肉生长发育过程中发挥重要的作用。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 2556 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 ofα-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 development of Tibetan sheep.

关 键 词：藏绵羊 PFKM基因 克隆 生物信息学分析 组织表达 

分 类 号：S826.2[农业科学—畜牧学]