水稻花药特异表达启动子的克隆与活性检测

Cloning and Activity Assaying of Anther-specific Promoter in Rice

植物雄性器官特异表达启动子的克隆是作物杂种优势利用分子育种的基础.根据水稻花药特异表达RA8基因序列设计引物,从水稻(Oryza sativaL.)品种日本晴中克隆得到水稻花药特异表达基因RA8启动子Tsp2,该启动子为RA8基因翻译起始位点上游828bp的片段,具有启动子特征序列TATA盒TATAAATA和真核生物启动子特征序列CAAT框,与RA8启动子的核苷酸序列同源性为97%.利用该启动子与报告基因GFP构建植物表达载体p1304-rap,采用基因枪法转化烟草花药,通过GFP的瞬时表达证明该启动子具有花药特异启动子活性.

The isolation and the function analysis of male-specific promoter is basic for heterosis and molecular breeding. A putative promoter Tsp2 was amplified and cloned from rice （ Oryza sativa L. ） genomic DNA by polymerase chain reaction （PCR） with the primers designed according to the sequence of rice anther-specific gene RA8. Tsp2 was cloned into pMD18-T vector and sequenced. Sequence analysis showed that it had 828 bp in length and contained TATA box and CAAT box. Tsp2 had 97% identity with RA8. Tsp2 was fused into reporter gene GFP and constructed to plant expression vector p1304-rap. The expression vector p1304-rap was transformed into anther of tobacco by Particle Bombardment. The transient expression of GFP was observed in tapetum particularly. No GFP expressed in leaf and pistil as control. The results in tobacco （ Nicotiana tobacum） anther indicated the activities of the promoter. The Tsp2 promotor fragment was cloned from rice genome, However it could work well in tobacco. Therefore, the Tsp2 promoter should be used widely in heterosis and molecular breeding of all dicot and monocot crops.