Journal of Nuclear Agricultural Sciences ›› 2017, Vol. 31 ›› Issue (7): 1282-1289.DOI: 10.11869/j.issn.100-8551.2017.07.1282

• Induced Mutations for Plant Breeding·Agricultural Biotechnology • Previous Articles     Next Articles

Cloning and Deletion Analysis of NtMTPC4 Promoter From Nicotiana tabacum

LIU Jikai1, 2, *, ZHANG Lin1, GAO Yongfeng1, WU Chanjuan1, 2, TANG Yunlai1, 2   

  1. 1 School of Life Science and Engineering, Southwest University of Science and Technology, Mianyang, Sichuan 621010;
    2 Fundamental Science on Nuclear Wastes and Environmental Safety Laboratory, Southwest University of Science and Technology, Mianyang, Sichuan 621010
  • Received:2016-08-03 Online:2017-07-10 Published:2017-05-11

烟草NtMTPC4启动子的克隆与缺失分析

刘继恺1, 2, *, 张林1, 高永峰1, 吴婵娟1, 2, 唐运来1, 2   

  1. 1 西南科技大学生命科学与工程学院,四川 绵阳 621010;
    2 西南科技大学核废物与环境安全国防重点实验室,四川 绵阳 621010
  • 通讯作者: 同第一作者。
  • 作者简介:刘继恺,女,讲师,主要从事植物分子生物学研究。E-mail:kateryan@163.com
  • 基金资助:
    国家自然科学基金(31500205),西南科技大学博士研究基金(14zx7153),四川省生物质资源利用与改性工程中心项目(13zxsk02)

Abstract: Tissue-specific promoters are important tools in genetic engineering and practical application. In order to study the expression characteristics of NtMTPC4 promoter from Nicotiana tabacum, the relative expression of NtMTPC4 was determined by quantitative real-time PCR (qRT-PCR). A 2 287 bp length promoter sequence of NtMTPC4 gene was cloned by PCR method. Based on the distribution of the regulatory elements, three truncations of NtMTPC4-P were obtained. The full length NtMTPC4-P and the truncations were then inserted into pBI121K respectively to replace the CaMV35S promoter. All the recombinant plasmids were introduced into Arabidopsis by Agrobacterium-tumefaciens mediated floral dipping method. qRT-PCR analysis indicated that NtMTPC4 gene was highly expressed in flowers. Sequence analysis showed that NtMTPC4-P contained basic cis-elements, such as CAAT-box, TATA-box and tissue specific regulation elements, GTGANTG10 and POLLEN1LELAT52. GUS histochemical assay showed that the expression of the GUS in all of the transgenic plants was detected particularly in pollens. These results provide a new regulatory element for the expression of target gene specifically in pollen by genetic engineering.

Key words: Nicotiana tabacum, NtMTPC4 promoter, pollen-specific promoter, cloning, deletion analysis

摘要: 组织特异性启动子是基因工程研究及实际应用中的重要工具。为了研究烟草NtMTPC4启动子的表达特性,采用实时荧光定量PCR方法分析烟草金属耐受蛋白C4(MTPC4)基因NtMTPC4在烟草不同组织中的表达模式,并利用PCR技术克隆得到NtMTPC4的上游启动子NtMTPC4-P。根据调控元件的分布对NtMTPC4-P进行不同程度的缺失,获得了NtMTPC4-P1、NtMTPC4-P2和NtMTPC4-P3缺失体;构建全长NtMTPC4-P和不同缺失体的植物表达载体,并通过花序浸染法转化拟南芥,对获得的转基因拟南芥进行GUS组织化学染色分析。结果表明,NtMTPC4基因的表达具有组织特异性,其在花中的表达量最高。NtMTPC4-P全长2 287 bp,含有CAAT-box、TATA-box、GTGANTG10和POLLEN1LELAT52等顺式作用元件和调控元件。GUS组织化学染色结果表明,全长启动子NtMTPC4-P和不同缺失体都能特异性地驱动GUS基因在转基因拟南芥花粉中的表达。本研究结果为通过基因工程技术在花粉中特异表达目的基因提供了新的调控元件。

关键词: 烟草, NtMTPC4启动子, 花粉特异性启动子, 克隆, 缺失分析