大豆脂肪氧化酶-3基因(Lox3)启动子的克隆及其瞬时表达分析

Cloning and Transient Expression of Soybean Lox3 Gene Promoter

利用PCR技术从大豆基因组DNA中分离脂肪氧化酶-3基因启动子片段Lox3p。PLACE在线启动子预测工具分析表明:序列中含有多种典型的种子及胚特异性表达元件。将克隆得到的Lox3p片段替换pCAMBIA1301中的CaMV35S启动子,构建表达载体pCAM-Lox3p。通过农杆菌介导法在大豆种子中进行瞬时表达,GUS组织化学染色及荧光测定都显示出Lox3p驱动GUS基因表达的强度高于CaMV35S启动子。结果表明,该Lox3p启动子片段具备一定的胚特异表达特性,为探明大豆脂肪氧化酶-3基因启动子胚特异表达调控序列及其调控机制的研究奠定基础。

The upstream nucleotide sequence of soybean Lox3 gene,named Lox3p,was isolated from the genomic DNA of soybean by PCR method.Promoter sequence analysis by PLACE showed that it had some typical seed-specific and embryo-specific cis-elements.Replacing CaMV35S promoter of pCAMBIA1301 with the Lox3p fragment,the binary expression vector pCAM-Lox3p was constructed.Transient expression in soybean seed by Agrobacterium tumefaciens mediated method,both the results of histochemical GUS analysis and fluorometric GUS analysis showed that GUS activity driven by Lox3p fragment was higher than that driven by CaMV35S promoter.The results indicated that the fragment of Lox3p might be similar or have retained certain embryo-specific features,which provided valuable information of supporting the future research of regulatory sequence and regulatory mechanism of Lox3 gene promoter.