獐茅高亲和性K+转运蛋白基因(AlHAK1)是从单子叶禾本科盐生植物獐茅(Aeluropus littoralis (Gouan) Parl)中克隆, 对于细胞营养和离子渗透调节起关键作用。为了进一步了解AlHAK1基因的表达调控机制, 文章采用基因组步移法分离了AlHAK1基因转录起始位点上游长度约1.3 kb的启动子区域。启动子顺式元件分析显示该序列具有典型的TATA和CAAT盒, 以及一些与植物生长发育和环境响应相关的顺式元件。为了明确AlHAK1启动子的功能, 将其与GUS基因融合构建到植物表达载体pCAMBIA1301上, 通过农杆菌介导转化法导入水稻中。对转基因植株进行GUS组织化学染色, 结果显示在转化AlHAK1启动子水稻的根、茎、叶、花药和内外稃部位均检测到GUS活性。GUS荧光定量分析显示AlHAK1启动子调节GUS表达活性低于组成型启动子CaMV35S和Ubiquitin, 但其根部和茎部的GUS活性相对较高。对转化植株进行不同胁迫处理后检测GUS活性, 结果表明受到ABA、干旱、高温的诱导后其茎部和根部GUS活性有所提高, 推测位于该启动子-682 bp的HSE元件和-1 268 bp的MybBS元件可能在高温、ABA和干旱诱导的表达调控中起作用。
The AlHAK1 gene encoding a high-affinity K+ transporter was isolated from Aeluropus littoralis (Gouan) Parl, a graminaceous halophyte, and plays a crucial role in nutrition and ion homeostasis in plant cell. To investigate the regulation role of AlHAK1 on the transcriptional level, an about 1.3 kb 5'-flanking region of the AlHAK1 gene containing a putative promoter was cloned by genome walking method. Cis-regulatory elements analysis showed AlHAK1-promoter region contained typical TATA and CAAT boxes, and some growth and development relative motifs, as well as environmental responsive elements. To reveal the function and regulating role, the Al-HAK1 promoter was fused to the β-glucuronidase (GUS) reporter gene in the pCAMBIA1301 vector and introduced into rice via Agrobacterium-mediated transformation. Histochemical staining indicated that the GUS expression directed by AlHAK1 promoter was observed in leaves, stems, roots, anther, lemma, and palea. GUS quantitative fluorometric analysis indicated that GUS activity directed by AlHAK1 promoter was lower than CaMV35S and Ubiquitin constitutive promoters; however, in the roots and stems the GUS activity was relatively high and displayed a tissue-specific expression pattern. Under ABA, high temperature or drought stress, the GUS activity directed by AlHAK1 promoter was inducible in the roots and stems, suggesting the elements of HSE (-682 bp) and MybBS (-1 268 bp) might play a role in the inducible regulation.
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