丹参SmPAP1基因组DNA的克隆及其拷贝数的验定

Cloning, Characterization of the Full-length Genomic Sequence of SmPAP1 Gene from Salvia miltiorrhiza and Determination of Gene Copy

本研究以获得的丹参SmPAP1基因c DNA为模板设计引物扩增其基因组DNA序列(genomic DNA,g DNA)。g DNA与c DNA序列比对结果显示,该基因基因组DNA序列长度为739 bp,由2个外显子和1个内含子组成。进一步采用染色体步移技术克隆了其上游的一段长度为1 197 bp的启动子序列。启动子序列分析结果显示,该区域内不仅含有保守的TATA盒和CAAT盒,而且包含多个潜在的与胁迫应答相关的顺式作用元件,暗示SmPAP1基因很可能响应多种环境胁迫诱导表达。此外,SmPAP1基因的Southern杂交结果表明,在丹参基因组中SmPAP1仅有1个拷贝数。本研究结果为进一步研究SmPAP1基因的表达调控模式以及启动子的功能奠定基础。

Based on the SmPAP1 c DNA sequence, genomic DNA of SmPAP1 corresponding to its coding region was cloned. The result of sequence alignment showed that SmPAP1 genomic DNA was 739 bp in length and contained two extons and one intron compared with full-length c DNA. A 1 179 bp 5'-flanking region of the SmPAP1 was further isolated from Salvia miltiorrhiza using genome walking method. The result of sequence analysis revealed that this fragment contained the conserved TATA and CAAT boxes upstream of the start codon,besides, several potential cis-acting elements associated with plant hormone and stress-related responses were located within the SmPAP1 promoter, indicating that this promoter might be a multiple stress-inducible one.Southern blotting result revealed that there was one copy of SmPAP1 in Salvia miltiorrhiza genome. The results of this study lay the foundation for further exploring expression regulation as well as promoter functional characterization of SmPAP1 gene.