柑橘CsHB1启动子的克隆及功能分析

以‘伏令夏橙’叶片基因组DNA为模板,克隆了CsHB1启动子5′端转录起始位点上游分别为1 741 bp和1 613 bp的区域序列。序列分析表明这两个启动子区序列相似度为90.7%,存在141 bp的差异片段,该差异片段中含有2个Box4元件。通过PlantCare数据库对启动子序列的顺式作用元件进行预测,结果表明CsHB1的不同启动子序列含有多种响应元件,如胚乳特异性元件、分生组织表达元件、光反应元件、热应激反应元件、MYB结合位点等。为进一步分析CsHB1启动子的功能,构建该基因启动子与GUS基因融合的植物表达载体prCsHB1::GUS,并用农杆菌介导法转化拟南芥,获得prCs HB1-1::GUS拟南芥纯合材料13株和prCs HB1-2::GUS拟南芥纯合材料10株。对转基因拟南芥进行GUS组织化学染色鉴定,结果表明两类启动子表达载体已成功转入拟南芥中并进行了表达,其表达部位主要集中于愈伤组织和花药中,而在种荚、叶片及幼苗中未检测到GUS蛋白的表达。

Isolation of the Citrus CsHB1 Gene Promoter and Its Preliminary Functional Analysis

Two promoter sequences（1 741 bp and 1 613 bp）of the somatic embryogenesis development-related gene CsHB1 were cloned from genomic DNA of‘Valencia’sweet orange. The sequence similarity of these two promoters is 90.7%. There is a 141 bp fragment difference between them，which contains two Box4 cis-acting elements. The cis-acting elements of promoter were analyzed and predicted using PlantCare databases. The results indicated that the CsHB1 promoter sequence included a variety of response elements，such as endosperm specific elements，meristem expression elements，light-responsive elements，heat stress responsiveness elements，and MYB binding site. In order to study the function of CsHB1 promoter，a promoter-reporter vector prCsHB1::GUS was constructed，and introduced into Arabidopsis by Agrobacterium-mediated method. A total of 13 homozygotes were obtained with prCsHB1-1::GUS，and a total of 10 homozygotes were obtained with prCsHB1-2::GUS. GUS staining activity was detected in different tissues. The results showed that two promoter vectors were transferred and expressed successfully in Arabidopsis，and the GUS activity was mainly detected in callus and anther，not in the pods，blade and seedlings.