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植物研究 ›› 2021, Vol. 41 ›› Issue (5): 729-737.doi: 10.7525/j.issn.1673-5102.2021.05.011

• 研究报告 • 上一篇    

梁山慈竹DfMYB3基因克隆及启动子分析

杜恬恬1, 李会萍1, 王博雅1, 欧倩1, 黄艳1, 曹颖1, 胡尚连1,2()   

  1. 1.西南科技大学植物细胞工程实验室,绵阳 621010
    2.四川省生物质资源利用与改性工程技术研究中心,绵阳 621010
  • 收稿日期:2020-12-29 出版日期:2021-09-20 发布日期:2021-07-05
  • 通讯作者: 胡尚连 E-mail:hushanglian@126.com;hashanglian@126.com
  • 作者简介:杜恬恬(1995—),女,硕士研究生,主要从事生物化学与分子生物学研究。
  • 基金资助:
    四川省应用基础研究项目(18YYJC0920);四川省“十三五”重点攻关项目(2016NYZ0038);四川省重点研发项目(2019YFN0005);环境友好能源材料国家重点实验室项目(19fksy0113)

Cloning and Promotor Analysis of DfMYB3 from Dendrocalamus farinosus

Tian-Tian DU1, Hui-Ping LI1, Bo-Ya WANG1, Qian OU1, Yan HUANG1, Ying CAO1, Shang-Lian HU1,2()   

  1. 1.Lab of Plant Cell Engineering Southwest University of Science and Technology,Mianyang 621010
    2.Engineering Research Center for Biomass Resource Utilizaiton and Modification of Sichuan Province,Mianyang 621010
  • Received:2020-12-29 Online:2021-09-20 Published:2021-07-05
  • Contact: Shang-Lian HU E-mail:hushanglian@126.com;hashanglian@126.com
  • About author:DU Tian-Tian(1995—),female,master,focused on biochemistry and molecular biology.
  • Supported by:
    Applied Basic Research Project of Sichuan Province(18YYJC0920);13th Five-Year Key Project of Sichuan Province(2016NYZ0038);Key Research and Development Projects of Sichuan Province(2019YFN0005);Environmentally friendly Energy Materials of State Key Laboratory Project(19fksy0113)

摘要:

基于梁山慈竹转录组数据库,以梁山慈竹叶片为材料,克隆得到一个MYB转录因子,命名为DfMYB3,其开放阅读框长度为1 287 bp,编码428个氨基酸,GenBank注册号为KY963358。保守结构域分析显示,DfMYB3有典型的SANT结构域,具有DNA-Binding结构域。系统进化树分析发现,DfMYB3与甘蔗、拟南芥、毛白杨等物种的R2R3-MYB转录因子聚集在一枝上。亚细胞定位结果显示,DfMYB3蛋白在细胞核以及细胞膜中均有表达,在细胞核上更为显著。通过染色体步移法克隆得到DfMYB3基因5′侧翼2 000 bp左右的序列。PlantCARE在线软件分析表明,该序列具有典型的启动子特征,并含有GA、ABA、MeJA等激素以及干旱等胁迫响应元件。100 μmol·L-1 GA、100 μmol·L-1 ABA处理梁山慈竹后,显著上调了DfMYB3基因的表达,表明DfMYB3基因能对GA及ABA处理产生应答。为探究DfMYB3启动子的功能,构建了DfMYB3启动子融合GUS基因的表达载体,并遗传转化烟草。在转基因烟草叶片及茎杆中都检测到了GUS信号,在叶脉处最为显著。

关键词: 梁山慈竹, MYB转录因子, 启动子, GA、ABA处理, GUS染色

Abstract:

Based on the transcriptome database of Dendrocalamus farinosus, a MYB transcription factor named DfMYB3 was cloned from the leave of D. farinosus. Its open reading frame length was 1 287 bp, encoding 428 amino acids, and GenBank accession Number was KY963358. The conserved domain analysis showed that DfMYB3 had the typical SANT domains and DNA-Binding domains. Phylogenetic tree analysis showed that DfMYB3 clustered with R2R3-MYB transcription factors in SaccharumArabidopsis and Populus. Subcellular localization showed that DfMYB3 protein was expressed in both the nucleus and cell membrane,and was more significant in the nucleus. The sequence of 5′ flanking of DfMYB3 gene about 2 000 bp was cloned by chromosome walking method. Analysis of PlantCARE online software showed that the sequence had typical promoter characteristics, and contained hormones responsive elements such as GA, ABA, MeJA as well as stress responsive elements such as drought. The expression level of DfMYB3 gene was significantly up-regulated after 100 μmol·L-1 GA and 100 μmol·L-1 ABA treatment, indicating that DfMYB3 gene was involved in response to GA and ABA treatments. In order to explore the function of DfMYB3 promoter, the expression vector of GUS gene fused with DfMYB3 promoter was constructed and genetically transformed into tobacco. The results showed that GUS signal was detected in both leaf and stem of transgenic tobacco, especially in the veins.

Key words: Dendrocalamus farinosus, MYB transcription factor, promotor, GA and ABA treatments, GUS staining

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