甘蓝型油菜RabGDI3基因启动子的克隆和功能验证

Cloning and function analysis of promoter of RabGDI3 from Brassica napus L.

以甘蓝型油菜(Brassica napus L.)基因组DNA为模板扩增获得BnRabGDI3基因翻译起始位点上游2 064bp的启动子序列。在线预测分析发现该启动子序列中有很多花药特异表达元件。将克隆得到的BnRabGDI3启动子取代pBI121中的CaMV35S启动子,构建BnRabGDI3启动子驱动报告基因gus表达的双元载体pBnRabG-DI3::GUS,并用根癌农杆菌介导法转化拟南芥。对获得的含有报告基因gus的6个转基因株系进行GUS活性分析。结果表明,在BnRabGDI3启动子的驱动下,报告基因gus仅在花药中特异表达。花药的冰冻切片显示gus仅在花药发育的第11到13期有表达,在花药的双核和三核期小孢子细胞中表达量最高。实验结果暗示BnRabGDI3基因可能与小孢子的发育相关。

Bioinformation showed that BnRabGDI3 gene promoter included a 2 064bp-long segment at the upstream translation start site in Brassica napus genome. Promoter sequence contained several anther specific cis-elements. To verify the biology function, a recombinant vector was designated as pBnRabGDI3：：GUS through the replacement of CaMV35S promoter in pBI121 by cloned BnRabGDI3 promoter fragment. In the vector, gus reporter gene was driven by BnRabGDI3 promoter. GUS histochemical assay in six independent transgenic plants showed that gus expressed only in anthers from the 11th to 13th development stages. The highest level of GUS activity was detected in bicellular and tricellar microspores, suggesting BnRabGDI3 played an important role in microspore development.