生物技术通报 ›› 2021, Vol. 37 ›› Issue (10): 17-25.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0077

• 研究报告 • 上一篇    下一篇

非洲菊GjMnSOD基因的克隆及表达分析

武欢1(), 卢珍红2,3, 郝向阳1, 王斌1, 焦元辰1, 杨春梅2,3, 程春振1,4()   

  1. 1.福建农林大学园艺学院,福州 350002
    2.云南省农业科学院花卉研究所,昆明 650100
    3.玉溪云星生物科技有限公司,玉溪 652604
    4.山西农业大学园艺学院,太谷 030801
  • 收稿日期:2021-01-18 出版日期:2021-10-26 发布日期:2021-11-12
  • 作者简介:武欢,女,硕士研究生,研究方向:花卉生物技术;E-mail: wuhuan980422@163.com;
  • 基金资助:
    福建省高原学科建设经费(102/71201801101);福建农林大学“校杰出青年科研人才”计划项目(xjq201721)

Cloning and Expression Analysis of GjMnSOD Gene in Gerbera jamesonni

WU Huan1(), LU Zhen-hong2,3, HAO Xiang-yang1, WANG Bin1, JIAO Yuan-chen1, YANG Chun-mei2,3, CHENG Chun-zhen1,4()   

  1. 1. College of Horticulture,Fujian Agriculture and Forestry University,Fuzhou 350002
    2. Flower Research Institute,Yunnan Academy of Agricultural Sciences,Kunming 650100
    3. Yuxi Yunxing Biotechnology Co.,Ltd.,Yuxi 652604
    4. College of Horticulture,Shanxi Agricultural University,Taigu 030801
  • Received:2021-01-18 Published:2021-10-26 Online:2021-11-12

摘要:

以非洲菊品种‘玲珑’为材料,利用RT-PCR技术克隆获得一条CDS长为519 bp的SOD基因(GenBank ID:MH708534.1)。利用生物信息学手段分析了该基因及其编码蛋白的序列特征,并利用实时荧光定量PCR(qRT-PCR)技术分析了该基因在3种激素(100 µmol/L IAA、SA和GA3)和3种非生物胁迫(200 mmol/L NaCl盐胁迫、30% PEG模拟干旱和4℃低温处理)下的表达模式。结果显示:该基因可编码分子量为19 203.85、等电点(pI)为7.93、无信号肽及跨膜结构的稳定亲水蛋白。该蛋白含有典型Sod-Fe-C保守结构域,在141-148位氨基酸之间含有一个MnSOD特征序列(DVWEHAYY),因此命名为GjMnSOD。Wolf Psort预测结果显示GjMnSOD定位于线粒体。GjMnSOD与刺苞菜蓟MnSOD相似性最高(为93.86%)且在进化上亲缘关系最近。qRT-PCR结果显示:GjMnSOD的表达受IAA、GA3、SA、盐胁迫和低温显著抑制;在干旱胁迫下,GjMnSOD的表达呈“先降后升”的表达趋势,于处理4 h后显著高于对照。研究结果表明GjMnSOD参与非洲菊对多种逆境胁迫的响应,尤其在非洲菊应答干旱胁迫过程中发挥重要作用。

关键词: 非洲菊, 锰超氧化物歧化酶, 基因克隆, 表达分析, 逆境胁迫响应

Abstract:

Having the ‘Linglong’ Gerbera jamesonni as experimental material,RT-PCR technology was used to successfully clone a SOD gene(GenBank accession number:MH708534.1)with CDS length of 519 bp. Then,bioinformatics analysis was to analyze the sequences of the gene and its encoded protein,and quantitative real time PCR(qRT-PCR)was adapted to investigate the gene expression patterns under treatments of 3 phytohormones(100 µmol/L IAA,GA3 and SA)and 3 abiotic stresses(salt stress using 200 mmol/L NaCl,mimicked drought stress using 30% PEG,and cold stress using 4℃). Results showed that the SOD gene encoded a stable hydrophilic protein with no signal peptide and transmembrane structure and its molecular weight was 19 203.85 and pI was 7.93. The protein contained typical Sod-Fe-C conserved domain and a MnSOD characteristic sequence(DVWEHAYY)ranging from 141 th to 148th amino acids,thus it was named as GjMnSOD. Wolf Psort prediction revealed that GjMnSOD was located in mitochondria. GjMnSOD shared the highest similarity(93.86%)with Cynara cardunculus MnSOD,and they were in the closest genetic relationship. qRT-PCR result showed that the expression of GjMnSOD was significantly inhibited by IAA,SA and GA3 treatments,salt and 4℃ low temperature stresses. Under drought treatment,it showed ‘fall-rise’ expression pattern and its expression level was found to be significantly higher than control from 4 h post-treatment. The above results indicate that GjMnSOD is widely involved in the stress responses of G. jamesonni,especially in drought response.

Key words: Gembera jamesonni, manganese superoxide dismutase, gene cloning, expression analysis, stress response