葡萄LEAFY基因启动子的克隆与序列分析

Cloning and sequence analysis of LEAFY gene promoter from grape(Vitis vinifera×V.labrusca)

为探明葡萄LEAFY基因的表达调控规律,应用PCR技术从藤稔葡萄中克隆了1个长1833 bp的DNA片段,该序列含有2个内含子区域,编码402个氨基酸,与葡萄LEAFY同源基因VFL有99%的同源性。应用基因组步移法克隆了LEAFY基因的5′侧翼序列925 bp,拼接后的LEAFY基因及启动子序列共2 692 bp(GenBank登录号EF222286)。用PLACE、P lantCARE在线启动子预测工具分析表明:该序列含有启动子的特定结构,如TATA-box,CAAT-box等,另外含有一些顺式作用元件如MYB结合位点、ABA响应元件、光响应元件和一些其他的调控序列,说明葡萄LEAFY基因的表达可能受MYB、ABA和光等的调控。用FootPrinter在线工具对葡萄与拟南芥等其他4种植物的LEAFY同源基因启动子进行比较,发现不同植物的启动子既有保守性,又有多样性,转录因子结合位点的分布相似,但也有区别,暗示了LEAFY基因表达调控的精确性或多样性。

In order to study the rule of grape LEAFY gene expression and regulation, a fragment from grape cultivar ‘Fujiminori＇ was amplified by PCR. It was 1 833 bp in length, deduced 402 amino acids, had two introns and 99% identity with grape LEAFY homologue VFL. 925 bp promoter region sequence was cloned by genome walking method. The gene and promoter were 2 692 bp in length （GenBank accession EF222286）. Promoter sequence analyzed by PLACE and PlantCARE showed that it had TATA-box, CAAT-box and some cis-acting element such as MYB binding site, cis-acting regulatory element involved in ABA response, some light-induced responsive elements and other transcription factor-binding sites. Compared the promoter of grape LEAFY homologue with other 4 plants by FootPrinter analysis, these promoter had conservatism and diversity and the distributing of transcription factorbinding sites had similarity and difference. It implied the accuracy and diversity of LEAFY gene expression and regulation.