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

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1800 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。

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 1800 bp and contained multiple cis-elements.The vectors Pro CiNAC038∶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 ABAinhibitive promoter.