水稻rbcs基因启动子的克隆及结构功能分析

利用PCR法克隆了水稻日本晴来源的核酮糖-1、5-二磷酸羧化酶小亚基基因（rbcS）5’上游调控区，命名为Posrbcs。将Posrbcs与gus基因融合，并通过农杆菌介导转入水稻中。对转基因水稻植株中GUS活性进行定性与定量分析，结果表明，Posrbcs启动子可驱动gus基因在转基因水稻植株的叶片、叶鞘、茎杆及颖壳中特异性表达，在胚乳中不表达。然后构建不同长度片断的Posrbcs的5’端缺失体，分别与gus构建融合基因，转入水稻中。对转基因植株GUS活性定量分析结果显示， Posrbcs片段愈短，GUS活性愈低；进一步的光诱导试验结果显示，光能明显提高gus基因表达活性，并且随着Posrbcs片段缩短，Posrbcs中的I box、GT1结合位点、GATA box、T box 等光诱导相关元件的缺失会造成在不同时间段的光诱导活性降低以及光诱导表达时间后移。凝胶阻滞试验证实Posrbcs序列中的这些光诱导相关元件有相应的核蛋白的结合。

Cloning and Functional Analysis of the Rice rbcS Gene Promoter

The regulative sequence (Posrbcs) of ribulose-1 ,5-bisphosphate carboxylase small subunit( rbcS ) genes in rice was cloned by PCR amplification. The cloned Posrbcs was fused with gus gene, and then introduced into rice (Oryza sativa ssp. japonica) by Agrobacterium-mediated transformation. The result of GUS histochemical staining showed that gus gene driven by Posrbcs expressed in leaf, culm, sheath and glume,respectively, and not in endosperm. Also, 5' end difference length deletions of Posrbcs were fused with gus gene and the fused genes were transformed into rice,respectively. Quantitative analysis of GUS activity showed that the expression level of gus gene was reduced with the deletion of Posrbcs. The result of this experiment also showed that light induction could enhance GUS activity. Furthermore, the deletion of light inducible elements such as I box, GT1 binding factors, GATA box and T box could reduce GUS activity and postpone the light-induction time. Electrophoretic mobility shift assays showed that I box, GT1 factor, T box and GATA box could bind with the nuclear protein.