生物学杂志

生物学杂志 ›› 2019, Vol. 36 ›› Issue (6): 36-.doi: 10.3969/j.issn.2095-1736.2019.06.036

• 研究报告 • 上一篇    下一篇

异叶天南星凝集素基因的克隆及蛋白的结构性质分析 

  

  1. 1. 安徽中医药大学 药学院, 合肥 230012； 2. 安徽中医药大学 研究生院, 合肥 230012；3. 安徽中医药大学科研实验中心 新安医学教育部重点实验室, 合肥 230038； 4. 安徽道地中药材品质提升协同创新中心, 合肥 230012； 5. 安徽省中医药科学院, 合肥 230012
  • 出版日期:2019-12-18 发布日期:2019-12-12
  • 通讯作者: 吴家文，教授，研究方向为中草药分子生物学，E-mail:wujiawen@ahtcm.edu.cn
  • 作者简介:李晟，研究方向为中草药分子生物学，E-mail:1666341819@qq.com
  • 基金资助:

    安徽省自然科学基金项目（1608085MH177）；安徽高校自然科学研究重大项目 （KJ2018ZD028）；名贵中药资源可持续利用能力建设项目（2060302）；大学生创新创业训练计划项目


Cloning and characterization of a lectin from Arisaema heterophyllum Blume 

  1. 1. School of Pharmacy, Anhui University of Traditional Chinese Medicine, Hefei 230012; 2. Graduate School, Anhui University of Traditional Chinese Medicine, Hefei 230012; 3. Key Laboratory of Xin′an Medicine, Ministry of Education, Anhui University of Traditional Chinese Medicine, Hefei 230038; 4. Synergetic Innovation Center of Anhui Authentic Chinese Medicine Quality Improvement, Hefei 230012; 5.Anhui Academy of Chinese Medicine, Hefei 230012, China
  • Online:2019-12-18 Published:2019-12-12

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摘要：

克隆了中药材异叶天南星（Arisaema heterophyllum Blume）叶中凝集素（lectin）基因（Ahltn）的开放阅读框（ORF），并对其编码蛋白AhLTN的性质和结构进行了分析。首先从植物异叶天南星的新鲜叶片中提取总RNA，通过逆转录获取cDNA，经PCR扩增Ahltn后与pMD19-T载体连接构成重组质粒，然后转化到大肠杆菌感受态细胞中培养，经菌液PCR鉴定后再进行测序验证，确定目的基因被成功克隆，进一步运用各种生物信息学软件对AhLTN的理化性质、进化关系和空间结构进行分析。结果显示，克隆获得了Ahltn的ORF，此基因片段长度为561 bp，编码186个氨基酸的蛋白质，经生物信息学分析AhLTN属于稳定蛋白，AhLTN与铁皮石斛的凝集素亲缘关系最近，四级结构显示其为二聚体，且两个单体的C末端发生了交换，每个单体中含有11个β折叠，呈“三棱镜”型，3个面均有保守的甘露糖结合区域。为天南星植物凝集素基因和蛋白功能的进一步研究和开发利用奠定了基础。


关键词: 异叶天南星, 凝集素, 基因克隆, 生物信息学分析

Abstract:

The open reading frame (ORF) of the lectin gene(Ahltn) was cloned from Arisaema heterophyllum Blume and the chief characteristic and structure of the coding protein was analyzed by bioinformatics tools. RNA was extracted from Arisaema heterophyllum Blume, and mRNA was purified and reverse-transcribed into cDNA. The target gene fragments were amplified by PCR and subcloned into pMD19-T vectors, and then transformed into E. coli competent cells. The recombinant plasmids were identified by PCR and further comfirmed by DNA sequencing. Moreover, we analyzed the physicochemical properties, homologous proteins and evolution, secondary structures and three dimensional structures of the AhLTN proteins by using bioinformatics softwares. The Ahltn full length ORF was successfully cloned, which was 561 base pair in length, encoding 186 amino acids. AhLTN was a stable dimer, which was assembled by “C-terminal β-sheet exchange”. Each subunit contained 11 β-sheets, showing a “a triangular prism” type. Three conserved tryptophan located in the center of the three dimensional structure, which constituted the hydrophobic region to keep the stable structure. Bioinformatics analysis showed that AhLTN had the closest relationship to that of Dendrobium catenatum. This paper will provide the basis for the further function study of Ahltn gene and AhLTN protein.


Key words: Arisaema heterophyllum Blume, lectin, gene cloning, bioinformatics analysis

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