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铁皮石斛DcCDPK8基因启动子克隆及功能分析

汪园1 高燕会1 朱玉球1 斯金平1

(1.浙江农林大学省部共建亚热带森林培育国家重点实验室国家林业局铁皮石斛工程技术研究中心, 浙江临安 311300)
【知识点链接】分子标记辅助育种

【摘要】铁皮石斛钙依赖性蛋白激酶基因Dc CDPK8参与低温激素ABA和MeJA的信号转导过程, 但转录调控尚不明确, 为了研究铁皮石斛Dc CDPK8基因的核心启动子区域, 并对其转录调控机制进行探讨, 采用PCR技术克隆铁皮石斛Dc CDPK8基因启动子序列, 并利用5'端缺失和GUS基因的融合表达研究启动子序列功能。结果表明Dc CDPK8基因启动子序列含有1个低温响应元件LTR (-1 749-614 bp) , 2个茉莉酸甲酯响应元件 (-1 749-230 bp) 和1个ABA响应元件 (-614-230 bp) , 构建3个5'端不同缺失片段融合GUS报告基因的p BI121真核表达载体, 瞬时转化烟草叶片, 经低温胁迫后GUS活性强弱为Dc CDPK8-p1>Dc CDPK8-p2>Dc CDPK8-p3, 经外源ABA诱导后GUS活性Dc CDPK8-p1>Dc CDPK8-p2>Dc CDPK8-p3, 而经外源MeJA诱导后GUS活性Dc CDPK8-p1>Dc CDPK8-p2>Dc CDPK8-p3, 推测Dc CDPK8基因启动子序列中ABA响应元件 (ARE) 对外源ABA的响应起正调控作用, 而MeJA顺式作用元件对外源茉莉酸甲酯响应起抑制作用。

【关键词】 铁皮石斛;DcCDPK8启动子;瞬时转染;组织化学染色;

【DOI】

【基金资助】 国家重点研发计划项目 (2017YFC1702201) ; 国家自然科学基金项目 (81603228) ;

Cloning and function analysis of promoter of DcCDPK8 from Dendrobium catenatum

WANG Yuan1 GAO Yan-hui1 ZHU Yu-qiu1 SI Jin-ping

(1.Dendrobium State Forestry Engineering Research Center, State Key Laboratory of Subtropical Silviculture, Zhejiang A & F University, Lin’an, Zhejiang Province, China 311300)

【Abstract】 DcCDPK8 is involved in abiotic stress such as low temperature stress and signal transduction of hormones ABA and MeJA, but the transcriptional regulation is still unclear. In order to study the core promoter region of DcCDPK8 gene in Dendrobium catenatum and explore its transcriptional regulation mechanism, the DcCDPK8 gene promoter sequence was cloned by PCR from D.catenatum. Promoter sequence function was studied by fusion of 5' terminal deletion and GUS gene. The results showed that the promoter sequence of DcCDPK8 gene has a low-temperature responsive element (LTR) between −1 749 bp and −614 bp, two MeJA responsive elements between −1 749 bp and −230 bp, and one ABA responsive element between −614 bp and −230 bp. Three 5'-end different deletion fragments were constructed to fuse the eukaryotic expression vectors p BI121 with GUS, which were transformed into tobacco leaves. The GUS activities under cold stress treatment were DcCDPK8-p1 > DcCDPK8-p2 > DcCDPK8-p3. GUS activities under exogenous ABA induction were DcCDPK8-p1 > DcCDPK8-p2 > DcCDPK8-p3, and GUS activities under exogenous MeJA induction were DcCDPK8-p1 > DcCDPK8-p2 > DcCDPK8-p3. It is speculated that the ABA response element (ARE) in the promoter sequences of DcCDPK8 plays a positive regulatory role in response to exogenous ABA, while the MeJA cis-acting element plays a negative role in response to exogenous MeJA.

【Keywords】 Dendrobium catenatum; DcCDPK8 promoter; transient transfection; histochemical staining;

【DOI】

【Funds】 National Key Research and Development Program (2017YFC1702201); National Natural Science Foundation of China (81603228);

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This Article

ISSN:1001-5302

CN: 11-2272/R

Vol 44, No. 02, Pages 293-297

January 2019

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Abstract

  • 1 Materials and methods
  • 2 Results and analysis
  • 3 Conclusion and discussion
  • References