小金海棠Fe3+-螯合还原酶MxFRO2基因启动子的克隆与表达分析

通过染色体步移法从小金海棠（Malus xiaojinensis）基因组中克隆了三价铁螯合还原酶（ferric chelate reductase, MxFRO2）基因翻译起始位点上游1 738 bp的启动子序列。生物信息学分析表明，该启动子片段中存在光响应元件G-box、生长素应答元件、铜元素响应元件、TATA-box、CAAT-box等顺式作用元件。从TAIR网站获得了拟南芥（Arabidopsis thaliana）中8个FRO基因上游的启动子序列，通过PlantCARE分析了其与MxFRO2启动子的异同。根据在线预测结果，克隆了翻译起始位点上游1 644 bp（MxFul）和259 bp（MxD1）两段序列，构建了其与GFP融合的瞬时表达载体，并将重组质粒通过PEG介导法分别转化拟南芥叶片原生质体，结果表明，小金海棠MxFul和MxD1两段启动子序列都能驱动GFP蛋白在拟南芥原生质体中的瞬时表达。

Cloning and Expression Analysis of Ferric Chelate Reductase MxFRO2 Gene Promoter from Malus xiaojinensis

The regulative sequence(1 738 bp) of ferric chelate reductase (MxFRO2) gene from Malus xiaojinensis was cloned by genomic walking. In silico analysis of sequence suggested that the sequence contained several typical cis-acting elements, including multiple light responsive elements (LREs), ABA responsive element (ABRE), auxin inducible element, Cu responsive element (CuRE), TATA-box and CAAT-box. The promoter sequences of 8 members of Arabidopsis FRO gene family gained from TAIR website were predicted as well. A 1 644 bp and a 259 bp promoter sequence upstream 5' of translation start site of MxFRO2 were cloned and designated as MxFul and MxD1, respectively. MxFul-GFP and MxFul-GFP were constructed and transformed into Arabidopsis mesophyll protoplasts mediated by PEG. These results showed that MxFul and MxD1 could both drive the transient expression of GFP protein in Arabidopsis mesophyll protoplasts.