柽柳GRAS转录因子基因启动子克隆和表达分析

doi:10.11924/j.issn.1000-6850.casb15080006

中国农学通报 ›› 2016, Vol. 32 ›› Issue (2): 28-32.doi: 10.11924/j.issn.1000-6850.casb15080006

所属专题：生物技术

柽柳GRAS转录因子基因启动子克隆和表达分析

李雪燕,金胶胶,赵玉琳,李冠霖,王培龙,高彩球

东北林业大学,东北林业大学,东北林业大学,东北林业大学,东北林业大学,东北林业大学国家级林木遗传育种实验室

收稿日期:2015-08-02 修回日期:2015-09-14 接受日期:2015-09-24 出版日期:2016-01-28 发布日期:2016-01-28
通讯作者: 高彩球
基金资助:
国家自然科学基金“柽柳Dof转录因子的耐盐调控机理研究”(31370676)；大学生创新实验训练项目国家级(201410225068)。

Cloning and Expression Analysis of Promoter of GRAS Transcription Factor from Tamarix hispida

Received:2015-08-02 Revised:2015-09-14 Accepted:2015-09-24 Online:2016-01-28 Published:2016-01-28

摘要/Abstract

摘要： 克隆获得柽柳GRAS 转录因子基因启动子序列，并对其表达模式进行分析，从而初步探究GRAS转录因子基因的表达特征和功能。CTAB法提取刚毛柽柳基因组DNA，按照Genome Walking Kit 说明克隆GRAS 转录因子基因启动子序列，将克隆获得的GRAS 转录因子基因启动子序列定向替换pCAMB1301 载体上的35S启动子序列，构建融合表达载体，以驱动GUS 基因表达，瞬时侵染拟南芥后进行GUS 基因的染色。成功克隆获得刚毛柽柳936 bp 的GRAS 转录因子基因启动子序列。PLACE 和PlantCARE 数据库分析结果表明该启动子不仅包含启动子区的核心元件CAAT-box 和TATA-box，还含有多个与逆境应答有关的顺式调控元件。成功将GRAS 基因启动子序列定向置换pCAMBIA1301 的35S 启动子，构建重组载体PGRAS::GUS。瞬时转化拟南芥后GUS 染色，结果显示转基因拟南芥叶片被染色而根部着色较浅。初步表明克隆获得的GRAS 基因启动子具有启动子表达活性，其可能参与了柽柳的抗逆应答，为进一步分析该基因的抗逆功能和抗逆机制奠定了基础。

关键词: 经营模式, 经营模式, 土壤养分, 山核桃

Abstract: In order to explore the expression characteristics and functions of GRAS transcription factor gene in Tamarix hispida, the promoter sequence of GRAS transcription factor was cloned and its expression patterns were analyzed. The genomic DNA of T. hispida was extracted by using CTAB method. The promoter sequence of GRAS gene was cloned by using the Genome Walking Kit according to the instructions. The 35S promoter sequence of the pCAMBIA1301 expression vector was replaced by the GRAS promoter sequence to construct the fusion expression vectors and drove the expression of GUS gene. Then, the fusion expression vectors were transiently expressed in Arabidopsis thaliana and the transformed seedlings were stained by GUS. The 936 bp gene promoter of GRAS transcription factor was cloned from Tamarix hispida. The analysis results by using the PLACE and PlantCARE software suggested that the promoter sequences contained not only the core elements such as CAAT- box and TATA- box, but also some cis- element related to the stress response. The DNA recombinant plasmid PGRAS::GUS was constructed successfully by using the GRAS promoter sequence to replace the 35S promoter sequence of pCAMBIA1301, and was transiently transferred into Arabidopsis. The results of GUS staining showed that the leaves of transformed Arabidopsis were stained while the roots were stained slightly. These results indicated that the GRAS gene promoter sequence had promoter expression activity, and it might play a role in the stress response of T. hispida. The results provided the foundation for a comprehensive analysis of the stress response function and mechanisms of GRAS gene in T. hispida.

李雪燕,金胶胶,赵玉琳,李冠霖,王培龙,高彩球. 柽柳GRAS转录因子基因启动子克隆和表达分析[J]. 中国农学通报, 2016, 32(2): 28-32.

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柽柳GRAS转录因子基因启动子克隆和表达分析

Cloning and Expression Analysis of Promoter of GRAS Transcription Factor from Tamarix hispida

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