摘要
【目的】扩增努比亚山羊LIM结构域基因1(LIM domain gene 1,LMCD1)并进行生物信息学分析,构建真核表达载体并检测LMCD1基因的表达情况,为研究努比亚山羊LMCD1基因功能及探究LMCD1基因在山羊骨骼肌肉发育中的作用提供依据。【方法】从努比亚山羊背最长肌组织中提取总RNA,应用RT-PCR方法扩增LMCD1基因CDS区序列,并进行生物信息学分析;将LMCD1基因以同源重组的方式连接pEGFP-N1载体,经酶切、测序鉴定后重组阳性质粒命名为pEGFP-N1-LMCD1;将pEGFP-N1-LMCD1重组质粒转染至山羊骨骼肌卫星细胞,通过实时荧光定量PCR检测努比亚山羊LMCD1基因的表达情况。【结果】努比亚山羊LMCD1基因CDS区序列全长1092 bp,编码363个氨基酸。LMCD1蛋白分子式为C_(1775)H_(2818)N_(508)O_(533)S_(29),分子质量为40.73 ku。努比亚山羊LMCD1基因CDS区序列与山羊相似性最高(99.8%),与斑马鱼相似性最低(55.4%),与其他物种的相似性在87.0%~98.8%之间。LMCD1蛋白无信号肽,不存在跨膜结构域,为亲水性蛋白。通过构建努比亚山羊pEGFP-N1-LMCD1真核表达载体并转染至骨骼肌卫星细胞,过表达LMCD1基因,产生绿色荧光信号。【结论】试验成功扩增LMCD1基因CDS区序列,构建了pEGFP-N1-LMCD1真核表达载体,并分析了生物学功能,为后续开展LMCD1基因在山羊骨骼肌肉发育中的机制研究提供了理论基础。
【Objective】The purpose of this study was to amplify LIM domain gene 1(LMCD1)in Nubian goats and analyze using bioinformatics,construct eukaryotic expression vector,and detect the expression of LMCD1 gene,so as to provide a reference for studying the function of LMCD1 gene in Nubian goats and exploring the role of LMCD1 gene in skeletal muscle development of goats.【Method】Total RNA was extracted from the longissimus dorsi muscle in Nubian goats.The CDS region of LMCD1 gene in Nubian goats was amplified by RT-PCR and analyzed by bioinformatics,and LMCD1 gene was connected to pEGFP-N1 vector by homologous recombination.After enzyme digestion and sequencing identification,the positive plasmid was named pEGFP-N1-LMCD1 recombinant plasmid.pEGFP-N1-LMCD1 recombinant plasmid was transfected into skeletal muscle satellite cells in goats,the expression of LMCD1 gene in Nubian goats was detected by Real-time quantitative PCR.【Result】The total length of LMCD1 gene CDS in Nubian goats was 1092 bp,encoding 363 amino acids.The molecular formula of LMCD1 protein was C_(1775)H_(2818)N_(508)O_(533)S_(29),and the molecular weight was 40.73 ku.The sequence of LMCD1 gene CDS in Nubian goats had the highest similarity with Capra hircus(99.8%)and the lowest similarity with Danio rerio(55.4%),and the similarity with other species was 87.0%to 98.8%.LMCD1 protein had no signal peptide and transmembrane domain,and was a hydrophilic protein.pEGFP-N1-LMCD1 eukaryotic expression vector in Nubian goats was constructed and transfected into skeletal muscle satellite cells.LMCD1 gene was overexpressed to produce green fluorescence signal.【Conclusion】In this study,LMCD1 gene CDS in Nubian goats was amplified successfully,the pEGFP-N1-LMCD1 eukaryotic expression vector was constructed,and its biological function was analyzed,which provided a theoretical basis for the follow-up study of the mechanism of LMCD1 gene in skeletal muscle development of goats.
作者
邹剑伟
邹菊红
申玉建
韦一
张叁保
连子童
徐建建
宋颖
黄艳娜
韦英明
蒋钦杨
郑自华
ZOU Jianwei;ZOU Juhong;SHEN Yujian;WEI Yi;ZHANG Sanbao;LIAN Zitong;XU Jianjian;SONG Ying;HUANG Yanna;WEI Yingming;JIANG Qinyang;ZHENG Zihua(College of Animal Science and Technology,Guangxi University,Nanning 530004,China;Guangxi University Agriculture and Animal Husbandry Industry Development Research Institute,Nanning 530004,China)
出处
《中国畜牧兽医》
CAS
北大核心
2022年第6期2033-2042,共10页
China Animal Husbandry & Veterinary Medicine
基金
广西重点研发计划项目(桂科AB20297012、桂科AB19245010)
广西牛羊产业创新团队(nycytxgxcxtd-2021-09)
国家重点研发计划项目(2021YFD1100104)。
