RTS启动子与GUS的嵌合基因在转基因水稻花药中的专一性表达

Anther Specific Expression of the Chimeric Gene of RTS Promoter and Coding Region of β-Glucuronidase Gene in Transgenic Rice( Oryza sativa L.)

通过PCR扩增 ,从籼稻品种IR36中克隆了RTS启动子的DNA片段 ;序列分析表明 ,所克隆的DNA片段含 12 5 7个核苷酸 ,与Lee等 (1996 )报告的序列比较 ,核苷酸同源性为 97.1% ;将 - 12 2 8～ + 2 5的RTS启动子区段与GUS编码区组成的嵌合基因插入双元载体的T DNA中 ,经根癌农杆菌介导转化 ,得到转基因水稻植株。GUS活力检测表明 ,此嵌合基因不仅能在花药绒毡层 ,而且还在花药表皮细胞、药室内壁以及花粉中表达 ,而 - 783～ + 2 5的 5’上游区与GUS编码区组成的嵌合基因则未在转基因水稻的花药中检测到其表达。

To confirm the function of the promoter of RTS gene isolated from rice by Lee et al . (1996) and partially charaterized as a tapetum specific promoter, and to test the usefulness of this promoter in obtaining male sterile plants by genetic engineering, the upstream regulatory region of RTS gene was cloned by us from Oryza sativa subsp. indica IR36 and sequenced (Fig.1). Results show that it contained 1257 nucleotides and showed a sequence similarity of 97.1% with reported sequence. The fragment from -1228 ～+25 nucleotide was fused to a coding region of β glucuronidase ( GUS ) gene and introduced into rice plant by Agrobacterium mediated gene transfer. PCR analysis and Southern blot hybridization (Plate Ⅰ 1,2) of total DNA extracted from transgenic plants indicated that the chimeric GUS gene had been integrated into the rice genome. Histochemical analysis of GUS activity showed that the expression of GUS gene, was exclusively within the anther (including tapetum, epidermis, endothecium and pollen) of transgenic rice plants(Table 1, PlateⅠ 3～5), but no expression of GUS gene was detected when the coding region of GUS gene fused to the upstream regulatory region of RTS gene from -783 ～+25 nucleotide(Table 1).