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草业学报 ›› 2019, Vol. 28 ›› Issue (11): 159-167.DOI: 10.11686/cyxb2018755

• 研究论文 • 上一篇    下一篇

苦豆子赖氨酸脱羧酶基因启动子在拟南芥中的表达分析

陆姗姗1,2,3, 洪园淑1,2, 刘萍1,2,*   

  1. 1.宁夏优势特色作物现代分子育种重点实验室,宁夏 银川 750021;
    2.宁夏大学农学院,宁夏 银川 750021;
    3.青岛求实职业技术学院,山东 青岛 266109
  • 收稿日期:2018-11-28 出版日期:2019-11-20 发布日期:2019-11-20
  • 通讯作者: *. E-mail: liupnxdx@126.com
  • 作者简介:陆姗姗(1990-),女,山东青岛人,硕士。E-mail: 15709602594@163.com
  • 基金资助:
    国家自然科学基金(81760685)资助

Expression analysis of SaLDC promoter from Sophora alopecuroides in Arabidopsis thaliana

LU Shan-shan1,2,3, HONG Yuan-shu1,2, LIU Ping1,2,*   

  1. 1.Key Laboratory of Modern Molecular Breeding for Dominant and Special Crops in Ningxia, Yinchuan 750021, China;
    2.College of Agronomy, Ningxia University, Yinchuan 750021, China;
    3.Qingdao Qiushi College, Qingdao 266109, China
  • Received:2018-11-28 Online:2019-11-20 Published:2019-11-20
  • Contact: *. E-mail: liupnxdx@126.com

摘要: 赖氨酸脱羧酶(LDC)基因是苦豆子中氧化苦参碱(OMA)生物合成的第一个关键酶基因。在已有苦豆子赖氨酸脱羧酶基因(SaLDC)基础上克隆得到该基因上游1260 bp的启动子序列,GenBank登录号为KY038928,前期在苦豆子愈伤组织中的瞬时表达研究显示该启动子具有启动活性。生物信息学分析发现该启动子区域除了拥有启动子区的基本顺式作用元件TATA-box和CAAT-box外,还具有多个与光信号、逆境应答等相关的顺式作用元件。为进一步研究SaLDC启动子的功能,构建了该启动子与β-葡萄糖苷酸酶(GUS)报告基因融合的植物表达载体并通过农杆菌介导遗传转化拟南芥,同时对光诱导和聚乙二醇(PEG)胁迫的转基因拟南芥进行GUS活性染色和定量分析。结果显示,在T2代转基因拟南芥幼苗的不同生长阶段和成株的各组织器官中均可检测到GUS酶活性,且随幼苗生长时间的延长,叶片中的表达活性下降;在成株叶片和花萼中的表达活性强于根、茎、花瓣和角果。光和PEG胁迫均能诱导转基因拟南芥中GUS的表达;GUS酶活性定量测定显示,短时间的PEG胁迫(1~2 h)GUS酶活性显著上调(P<0.05),而连续胁迫8 h时GUS酶活性下调至最低(P<0.01),比胁迫前下降了28.2%。以上结果表明SaLDC启动子既有时空表达特异性又有组织表达特异性,光诱导和干旱胁迫对结构基因的表达有重要的调控作用。

关键词: 苦豆子, 赖氨酸脱羧酶, SaLDC启动子, 表达分析

Abstract: Lysine decarboxylase (LDC) gene is the first key enzyme gene in oxymatrine (OMA) biosynthesis in Sophora alopecuroides. The promoter sequence (1260 bp, gene bank accession number: KY038928) upstream of the gene (SaLDC) encoding lysine decarboxylase was cloned from S. alopecuroides genomic DNA. Its promoter activity was evaluated by transient expression in S. alopecuroides callus. A bioinformatics analysis showed that the promoter sequence contains a TATA-box, a CAAT-box, and other cis-acting elements such as drought-responsive, light-responsive, defense-responsive, stress-responsive elements, methyl jasmonate-responsive element, and tissue-specific expression element. To further study the functionality of the SaLDC promoter, a plant expression vector with the SaLDC promoter sequence fused to the GUS reporter gene (encoding β-glucuronidase) was transferred into Arabidopsis thaliana by Agrobacterium. β-glucuronidase activity was quantified by GUS staining and quantitative assays. We detected GUS activity in T2 transgenic A. thaliana seedlings at different growth stages and in tissues of the adult plants. During seedling growth, GUS activity in the leaves decreased. In adult plants, GUS activity was higher in the leaves and calyxes than in the roots, stems, petals and pods. The expression of GUS was induced by simulated drought (polyethylene glycol, PEG) stress and light in transgenic A. thaliana. Quantitative analyses showed that GUS activity increased significantly in the early stage of PEG stress (1-2 h) (P<0.05) and then decreased to a minimum at 8 h (P<0.01), to around 28.2% of its original level. All these results show that the SaLDC promoter confers tissue-specific and spatio-temporal-specific expression, and that the expression of this structural gene is regulated by light and drought stress.

Key words: Sophra alopecuroides, lysine decarboxylase (LDC), SaLDC promoter, expression analysis