Cloning and functional analysis of a promoter with temporal-spatial expressing differentiation in rice root | 一个水稻根时空差异表达启动子的克隆及功能分析

Chinese. 本研究旨在分离克隆水稻根特异表达的启动子，为育种提供资源。本实验结合本室芯片数据库和RT-PCR验证得到一个水稻根特异表达的基因(TIGR Locus：Loc_Os05g19570)，采用PCR技术从MH63的基因组DNA中扩增其上游启动子P12，长度为1 950 bp，将该启动子片段与报告基因gus相连，构建植物表达载体，农杆菌(Agrobaterium tumefaciens)介导转化水稻(Oryzative ssp. japonica)中花11。组织化学分析及荧光定量测定显示该启动子具有根特异性及时间差异性。根据P12启动子序列特征，利用PCR对其进行有目的的缺失，将5个长度不同的缺失片段分别与gus相连，构建植物表达载体，转化水稻中花11。荧光定量测定结果表明：－1 059~－819 bp区域缺失后，报告基因在根里的表达量显著降低，说明这个区域可能存在顺式元件。本研究成功的分离克隆到一个水稻根特异的启动子P12，进一步分析发现其还具有时间差异性。

English. This study aimed to isolate and clone root-specific promoters in rice and provide resources for breeding. Integrating of our lab’s database, a root specific gene (TIGR Locus: Loc_Os05g19570) was obtained after RT-PCR. A promoter named P12 with 1 950 bp in length of the gene was cloned by PCR from the genomic DNA of MH63. The cloned promoter was fused with gus gene to construct expression vector, and then introduced into rice (Oryza sativa ssp. japonica) Zhonghua11 by Agrobacterium-mediated transformation. The result of GUS histochemical staining and quantitative analysis showed that the promoter had root specificity and time difference. Also, 5' end different length deletions of P12 were fused with gus gene and were transformed into rice, respectively. Quantitative analysis of GUS activity showed the expression level of gus gene in root was badly reduced after deletion the region of －1 059~－819 bp, it displayed some cis-elements maybe presence in this region. A root-specific promoter was cloned in this study.