摘要
本试验旨在进行关岭牛肌球蛋白重链1(myosin heavy chain 1,MYH1)5′侧翼启动子的克隆和生物信息学分析。采集关岭牛血液及组织样品(背最长肌、后腿肌、心脏、肝脏、小肠、脂肪组织),利用PCR方法扩增关岭牛MYH1基因5′侧翼区,构建关岭牛pUCm-T-MYH1克隆载体,并对MYH1基因5′侧翼区进行生物信息学分析,最后利用实时荧光定量PCR法测定了MYH1基因在关岭牛不同组织中的表达。结果显示,本试验成功获得1 373bp(-1 360^+12bp)的MYH1基因5′侧翼启动子序列;利用生物信息软件对获得的克隆序列进行分析发现,MYH1基因5′侧翼启动子序列存在5处可能的转录起始点和多个潜在转录因子结合位点;关岭牛与金丝猴、野猪、家鼠、藏羚羊、野驴的MYH1基因5′侧翼区保守性较强的区域为转录起始点上游-400^+100bp,推测转录起始点上游-400^+75bp可能是其核心启动子区域。实时荧光定量PCR分析发现,MYH1基因在关岭牛背最长肌和后腿肌中高表达,在心脏、肝脏、小肠、脂肪组织中表达量很低,说明MYH1基因表达具有组织特异性。
The experiment was conducted to clone and analyze the sequence of MYH1 gene 5′-flanking region of Guanling cattle.The blood and tissues samples（longissimus dorsi,small intestine,hind leg muscles,heart,liver and adipose tissue）were collected,MYH1 gene 5′-flanking region of Guanling cattle was amplified by PCR,pUCm-T-MYH1 vector was constructed.The phylogenetic tree and promoter prediction was analysed by bioinformatics software,and the MYH1 gene expression in different tissues were compared by quantitative Real-time PCR.The results showed that 1 373 bp（-1 360 to＋12 bp）sequence of MYH1 gene 5′-flanking region of Guan-ling cattle was successfully amplified by PCR.The results of bioinformatics analysis showed that there were 5 transcription start sites and multiple potential transcription factor binding sites.The highly conserved region of Guanling cattle,Rhinopithecus roxellana,Sus scrofa,Mus musculus,Pantholops hodgsonii and Equus asinus was in-400 to＋100 bp of the upstream of transcription start sites,indicating that the-400 to＋75 bp of the upstream of transcription start sites was the core.The results of quantitative Real-time PCR showed that the expression of MYH1 gene in longissimus dorsi and hind leg muscles were higher,and that in other tissues were very low,indicating MYH1 gene had different expression in different tissues.
作者
陈伟
许厚强
周迪
张青青
赵焕平
王圆圆
CHEN Wei1,2,3,4 , XU Houqiang1,2,3, ZHOU Di1,2,3,4 , ZHANG Qingqing1,2,3 , ZHAO Huanping1,2,3 , WANG Yuanyuan1,2,3(1. Key Laboratory of Animal Genetics, Breeding and Reproduction in the Plateau Mountainous Region, Ministry of Education, Guiyang 550025, China ;2. Key Laboratory of Animal Genetics, Breeding and Reproduction in Guizhou, Guiyang 550025, China;3. College of Animal Science, Guizhou University, Guiyang 550025, China; 4. College of Life Science, Guizhou University, Guiyang 550025, Chin)
出处
《中国畜牧兽医》
CAS
北大核心
2018年第3期620-627,共8页
China Animal Husbandry & Veterinary Medicine
基金
国家自然科学基金青年科学项目(31401054)
国家自然科学基金常规面上项目(31571279)
黔科合重大专项(黔科合NY字[2013]6008号)
