中间锦鸡儿CiNAC038启动子的克隆及对激素响应分析

doi:10.13560/j.cnki.biotech.bull.1985.2019-1078

生物技术通报 ›› 2020, Vol. 36 ›› Issue (7): 55-61.doi: 10.13560/j.cnki.biotech.bull.1985.2019-1078

中间锦鸡儿CiNAC038启动子的克隆及对激素响应分析

红格日其其格¹, 王燕飞¹, 高仙灵², 庞彩霞², 尚晓蕊¹, 李国婧¹, 王瑞刚¹

1.内蒙古农业大学生命科学学院植物逆境生理与分子生物学自治区重点实验室,呼和浩特 010011;
2.呼和浩特职业学院,呼和浩特 010051

收稿日期:2019-11-06 出版日期:2020-07-26 发布日期:2020-07-28
作者简介:红格日其其格,女,博士研究生,研究方向：生物化学与分子生物学;E-mail：honggeriqiqige@126.com
基金资助:
国家自然科学基金项目（31660065）,内蒙古自然科学基金重大项目（2019ZD05）

Cloning of CiNAC038 Gene Promoter in Caragana intermedia and Profiling of Its Responses to Hormones

HONG Ge-riqiqige¹, WANG Yan-fei¹, GAO Xian-ling², PANG Cai-xia², SHANG Xiao-rui¹, LI Guo-jing¹, WANG Rui-gang¹

1. College of Life sciences,Inner mongolia Agricultural University,Inner Mongolia Key Laboratory of Plant Stress Physiology and Molecular Biology,Hohhot 010011;
2. Hohhot Vocational college,Hohhot 010051

Received:2019-11-06 Published:2020-07-26 Online:2020-07-28

摘要/Abstract

摘要： NAC转录因子家族是植物中特有的、家族数目较多的一类转录因子家族,对植物生长发育起重要作用。了解CiNAC038的表达调控分子机制,为中间锦鸡儿CiNAC038功能研究奠定基础。以中间锦鸡儿为植物材料,通过染色体步移法克隆启动子序列,并对启动子序列上的响应元件进行分析。构建GUS表达载体,并转化拟南芥,对拟南芥的组织特异性进行表达分析,用ABA诱导转基因植株,研究ABA与CiNAC038的关系。结果显示,克隆了1 800 bp的CiNAC038启动子序列,该启动子包含多种顺式元件。成功构建植物表达载体ProCiNAC038∶GUS,通过浸花法转化至野生型拟南芥。GUS组织化学染色结果显示,转基因拟南芥幼苗根部染色较深,胚轴无染色;成熟期转基因拟南芥的叶脉、果荚两端、花瓣、花药等组织染色较深,茎无染色。CiNAC038启动子驱动的GUS报告基因主要在植物叶片、根和花的组织器官表达。进一步ABA诱导表达分析发现,GUS染色随着浓度增加颜色越浅。CiNAC038启动子是ABA抑制型启动子。

关键词: 中间锦鸡儿, CiNAC038, 启动子, GUS染色

Abstract: The NAC transcription factor family is one of the large specific families in plants and play essential roles in plant development. To understand the molecular mechanism of CiNAC038 expression regulation would lay the foundation for the study of CiNAC038 function in Caragana intermedia. Genome walking method was used to clone the promoter sequence of CiNAC038 gene from genomic DNA of C. intermedia,and Cis-acting elements of the promoter were analyzed. The GUS expression vector was constructed and transformed into Arabidopsis thaliana,and the tissue-specific expression analysis of A. thaliana was performed. Transgenic plants were induced with ABA and the relationship between ABA and CiNAC038 was investigated. The result showed that the cloned fragment was 1 800 bp and contained multiple cis-elements. The vectors ProCiNAC038∶GUS was constructed successfully,and transformed into wild-type A. thaliana by floral dip method. The results of GUS histochemical staining showed that the roots of transgenic Arabidopsis seedlings were darkly stained while the hypocotyls were unstained. The mature veins,fruit pod ends,petals,anthers and other tissues of the transgenic Arabidopsis were darkly stained while the stems were unstained. The GUS reporter gene driven by the CiNAC038 promoter was mainly expressed in tissues and organs of plant leaves,roots and flowers. ABA-induced expression revealed that GUS stains became lighter with increasing concentration. CiNAC038 promoter might be an ABA-inhibitive promoter.

Key words: Caragana intermedia, CiNAC038, promoter, GUS staining

红格日其其格, 王燕飞, 高仙灵, 庞彩霞, 尚晓蕊, 李国婧, 王瑞刚. 中间锦鸡儿CiNAC038启动子的克隆及对激素响应分析[J]. 生物技术通报, 2020, 36(7): 55-61.

HONG Ge-riqiqige, WANG Yan-fei, GAO Xian-ling, PANG Cai-xia, SHANG Xiao-rui, LI Guo-jing, WANG Rui-gang. Cloning of CiNAC038 Gene Promoter in Caragana intermedia and Profiling of Its Responses to Hormones[J]. Biotechnology Bulletin, 2020, 36(7): 55-61.

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中间锦鸡儿CiNAC038启动子的克隆及对激素响应分析

Cloning of CiNAC038 Gene Promoter in Caragana intermedia and Profiling of Its Responses to Hormones

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