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作物学报 ›› 2015, Vol. 41 ›› Issue (04): 593-600.

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

受多逆境诱导表达的GhWRKY64基因启动子克隆与功能分析

杜皓,丁林云,何曼林,蔡彩平,郭旺珍*   

  1. 南京农业大学作物遗传与种质创新国家重点实验室 / 教育部杂交棉创制工程研究中心, 江苏南京210095
  • 收稿日期:2014-10-17 修回日期:2015-02-06 出版日期:2015-04-12 网络出版日期:2015-03-02
  • 通讯作者: 郭旺珍, E-mail: moelab@njau.edu.cn, Tel: 025-84396523
  • 基金资助:

    本研究由国家转基因生物新品种培育重大专项(2011ZX08005-004),江苏省农业科技自主创新资金项目[cx(14) 2065]和江苏省现代作物生产协同创新中心(JCIC-MCP)资助。

Cloning and Functional Identification of Promoter Region of GhWRKY64Induced by Multi-stresses in Cotton (Gossypium hirsutum)

DU Hao,DING Lin-Yun,HE Man-Lin,CAI Cai-Ping,GUO Wang-Zhen*   

  1. State Key Laboratory of Crop Genetics & Germplasm Enhancement, Hybrid Cotton Research & Development Engineering Research Center, Ministry of Education, Nanjing Agricultural University, Nanjing 210095, China
  • Received:2014-10-17 Revised:2015-02-06 Published:2015-04-12 Published online:2015-03-02
  • Contact: 郭旺珍, E-mail: moelab@njau.edu.cn, Tel: 025-84396523

摘要:

WRKY蛋白属于锌指型转录调控因子,参与植物生长发育及耐逆响应。以陆地棉遗传标准系TM-1为材料,克隆GhWRKY64(KF031101)基因上游1064bp的启动子序列,并对其调控元件及功能进行分析。生物信息学分析表明, 该区域含18个组织器官表达及诱导表达关键元件,分别为6个ROOTMOTIFTAPOX1根特异调控元件,4个CACTFTPPCA1叶肉特异性调控元件,4个OSE2ROOTNODULE病菌诱导元件,2个GTIGMSCAM4盐调控元件和2个W-box胁迫应答响应元件。将该启动子与GUS基因融合,构建pBIW64:GUS植物表达载体,通过农杆菌介导叶盘转化法获得12个转基因烟草株系。选择GUS表达量最高的pBIW64-5进行转基因不同组织器官表达及诱导表达分析。GUS组织化学染色显示,苗期的转基因烟草植株在叶和根部均具有GUS活性,开花期在转基因烟草植株根、叶及叶柄均检测到GUS活性,特别在转基因烟草的根及根尖部分染色更深,在茎和花组织上未检测到GUS活性。对该转基因烟草幼苗进行黄萎病菌诱导处理,与未处理的对照相比,诱导处理后的转基因烟草幼苗其根和叶片GUS染色明显加深。结果表明,GhWRKY64上游1064bp长度的DNA序列含根和叶组织优势表达启动子,且受病原菌诱导上调表达。该启动子可为开展棉花抗黄萎病转基因研究提供调控元件。

关键词: GhWRKY64启动子, 逆境胁迫, 转基因烟草, 调控元件, GUS活性

Abstract:

WRKY proteins are members of a transcription factor family with Zinc-finger structure in higher plant, which play diverse roles in plant responses to stresses and in various physiological processes.A 1064bp promoter sequence of GhWRKY64 was isolated from Gossypium hirsutum acc. TM-1, and its regulatory elements and functional characterization were further analyzed. Bioinformatics analysis showed that there were 18 putative tissue-speci?c or stress-induced regulatory motifs corresponding to known cis-elements in eukaryotic genes, including six ROOTMOTIFTAPOX1 of root-specific regulatory elements, two W-boxes, four CACTFTPPCA1mesophyll-specific regulatory elements, four OSE2ROOTNODULE of pathogenic bacteria-induced elements, and two GTIGMSCAM4 of salt regulatory elements. Further, pGhWRKY64 promoter region was fused to a β-glucuronidase(GUS) reporter gene as pBIW64:GUS constructed for investigation of important regions controlling gene expression, and 12 transgenic tobacco plants were obtained by Agrobacterium-mediated leaf-disc transformation method. Further, pBIW64-5 line with the highest GUS expression was selected for different tissues/organs expression and induced expression analyses. Histochemical staining in seedlings stage of the transgenic tobacco plants showed that the full-length promoter directed ef?cient expression of the reporter gene in the root and leaf, however, no GUS activity was detected in non-transgenic control.When transgenic tobacco plants growed at the flowering stage,GUS activity in root, leaf and petiole was detected, especially in root and root tip with heavier staining than those in other tissues, but no GUS activity was detected in stem and flower tissues. After treatment with Verticilliumdahliae, pBIW64:GUS showed greater induction and stronger GUS activity than those in untreated transgenic tobacco plants. Taken together, pGhWRKY64 promoter with 1064bp iselite regulatory element for preferential accumulation of foreign genes in root and leaf tissues and stress responses, which will be used for transgenic research in cottonVerticillium dahliae-resistant breeding.

Key words: GhWRKY64promoter, Abiotic and biotic stress, Transgenic tobacco, Regulatory elements, GUS activity

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