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园艺学报 ›› 2020, Vol. 47 ›› Issue (1): 85-97.doi: 10.16420/j.issn.0513-353x.2019-0091

• 研究论文 • 上一篇    下一篇

蝴蝶兰AP2/ERF家族基因的克隆及在低温下表达特性分析

崔 波1,*,郝平安2,梁 芳1,张 燕1,王喜蒙1,4,李俊霖1,3,蒋素华1,许申平1   

  1. 1郑州师范学院生物工程研究中心,郑州 450044;2西北农林科技大学生命科学学院,陕西杨凌 712100;3河南农业大学生命科学学院,郑州 450002;4郑州大学生命科学学院,郑州 450001
  • 出版日期:2020-01-25 发布日期:2020-01-25
  • 基金资助:
    郑州师范学院科技创新团队支持计划项目;河南省高等学校重点科研项目(18A180034,18B210015);河南省科技攻关计划项目(182102110369,182102110357)

Cloning and Expression Analysis of AP2/ERF Family Gene from Phalaenopsis Under Low Temperature

CUI Bo1,*,HAO Ping’an2,LIANG Fang1,ZHANG Yan1,WANG Ximeng1,4,LI Junlin1,3,JIANG Suhua1,and XU Shenping1   

  1. 1Biotechnology Research Center,Zhengzhou Normal University,Zhengzhou 450044,China;2College of Life Sciences,Northwest A & F University,Yangling,Shaanxi 712100,China;3College of Life Sciences,Henan Agricultural University,Zhengzhou 450002,China;4School of Life Sciences,Zhengzhou University,Zhengzhou 450001,China
  • Online:2020-01-25 Published:2020-01-25

摘要: 用RT-PCR方法从‘大辣椒’蝴蝶兰叶片中克隆得到4个AP2/ERF家族基因,命名为PhAP2/ERF1 ~ PhAP2/ERF4。蛋白结构域和序列比对发现PhAP2/ERF1 ~ PhAP2/ERF4均含有1个AP2结构域, PhAP2/ERF2还含有1个B3结构域。进化树分析表明4个蛋白与兰科AP2/ERF家族成员亲缘关系最近,PhAP2/ERF1属于AP2/ERF家族DREB亚家族中的A1类,PhAP2/ERF2属于RAV亚家族,PhAP2/ERF3属于ERF亚家族的B4类,PhAP2/ERF4属于DREB亚家族中的A2类。用实时荧光定量PCR方法检测低温驯化和低温胁迫条件下PhAP2/ERF1 ~ PhAP2/ERF4在‘大辣椒’和‘富乐夕阳’蝴蝶兰叶片中的表达特性,结果表明:4个基因在‘大辣椒’叶片中的表达趋势一致,表达量在低温胁迫早期达到最高,在胁迫中晚期有所下降;而4个基因在‘富乐夕阳’叶片中的表达趋势有较大差异,PhAP2/ERF1和PhAP2/ERF2的表达在低温处理整个过程中没有被诱导或仅有较小幅度增加,PhAP2/ERF3对低温的响应时间显著晚于‘大辣椒’,PhAP2/ERF4的表达趋势与‘大辣椒’差异较小。蝴蝶兰AP2/ERF在抗冷品种‘大辣椒’和不抗冷品种‘富乐夕阳’叶片中的表达差异暗示AP2/ERF基因家族在蝴蝶兰低温胁迫响应中起重要调控作用。

关键词: 蝴蝶兰, 低温胁迫, AP2/ERF基因, 基因克隆, 表达分析

Abstract: In this study,four AP2/ERF family genes named PhAP2/ERF1–PhAP2/ERF4 were cloned from the leaves of the Phalaenopsis‘Big Chili’by RT-PCR. Each PhAP2/ERF1–PhAP2/ERF4 contained an AP2 domain whereas the PhAP2/ERF2 also contained a B3 domain. The phylogenetic tree analysis showed that PhAP2/ERF1–PhAP2/ERF4 was closely related to members of AP2/ERF family in Orchidaceae. In addition,we found that the PhAP2/ERF1 belonged to the A1 group of DREB subfamily in AP2/ERF family,PhAP2/ERF2 belonged to RAV subfamily,PhAP2/ERF3 belonged to the B4 group of ERF subfamily,and PhAP2/ERF4 belonged to the A2 group of DREB subfamily. Real-time fluorescence quantitative PCR was used to investigate the expression levels of PhAP2/ERF1–PhAP2/ERF4 in the leaves of Phalaenopsis‘Big Chili’and Phalaenopsis‘Fuller’s Sunset’under low temperature acclimation and low temperature stress. The results showed that the expression of four PhAP2/ERF genes have the same trend in leaves of‘Big chili’,with the highest level at the early stage and decreased slightly at the middle and late stages after low temperature stress treatment. However,the expression levels of four PhAP2/ERF genes showed the significant differences trends in the leaves of Phalaenopsis‘Fuller’s Sunset’. The expression levels of PhAP2/ERF1 and PhAP2/ERF2 were not induced or increased slightly during the low temperature treatment. Moreover,the response time of PhAP2/ERF3 in leaves of Phalaenopsis‘Fuller’s Sunset’noticeably lagged behind that of Phalaenopsis‘Big Chili’under low temperature. There was little difference of PhAP2/ERF4 between Phalaenopsis‘Fuller’s Sunset’and Phalaenopsis‘Big Chili’. The difference in the expression of AP2/ERFs in the leaves between cold- resistant variety Phalaenopsis‘Big chili’and nonresistant variety Phalaenopsis‘Fuller’s Sunset’suggested that the AP2/ERF gene family of Phalaenopsis hybrida plays an important role in the regulation of low temperature stress response in Phalaenopsis species.

Key words: Phalaenopsis, low temperature stress, AP2/ERF gene, gene clone, expression analysis

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