关键词: 橡胶树, 启动子克隆, Adaptor-PCR, 方法改良, 应用实例

Abstract: Transcriptional regulation of eukaryotic genes is realized by the interactions between cis-elements and trans-acting factors. Most of the cis-elements reside in the promoter regions, and form the major factor controlling the start site and efficiency of transcription. Therefore, cloning and functional characterization of the promoter of a target gene becomes an important task in unraveling the mechanisms of gene expression and regulation. Here, an improved method of promoter cloning was established based on adaptor-PCR. We modified the adaptor sequences of the adaptor by increasing the specificity and annealing temperatures for the adaptor primers, and made it more suitable for two-step PCR. The genomic DNA of Rubber tree was cleaved by 25 selected restriction enzymes, then blunted and ligated to the same modified adaptor, and formed the genome-walking library for promoter cloning. The utility of this library was verified by the successful promoter cloning for six sucrose transporter genes, one invertase gene and one trehalose synthase gene. The method established in this work is suitable for promoter cloning in Rubber tree as well as other plant species.

Key words: Hevea brasiliensis, promoter cloning, adaptor-PCR, method improvement, application