摘要
Previously, a moderately repetitive DNA sequence （RRD3） was cloned from rice （Oryza sativa L.） by DNA renaturation kinetics. Sequence analysis revealed several conserved promoter motifs, including four TATA-boxes and a CAAT-box, and promoter activity was shown in Escherichia coli and mammalian expression systems. Here, we inserted the RRD3 fragment into the plant promoter-capture vector, pCAMBIA1391Z, and examined whether the RRD3 fragment has promoter activity in plants. Transgenic tobacco and rice calli both showed β-glucuronidase （GUS） activity, indicating that RRD3 can act as a promoter in both monocot and dicot plants. Based on the promoter characteristic of RRD3, we designed a plant universal binary vector, pCRiRRD3, which is suitable for performing researches on plant RNA interference. This vector has two multiple cloning sites to facilitate sense and antisense cloning of the target sequence, separated by an intron fragment of 200 bp. The efficiency of the vector for gene silencing was assayed by histochemical and quantitative fluorometric GUS assays in transgenic tobacco. These research results suggested that this plant RNAi vector pCRiRRD3 can effectively perform gene silencing researches on both monocot and dicot plants.
RRD3是通过复性动力学从水稻基因组中克隆到的一个中度重复序列,序列分析揭示其含有多个保守的启动子元件,包括4个TATA-boxes和CAAT-box,在Escherichia coli及哺乳动物表达系统中均表现出启动子活性。我们将RRD3片段插入到植物启动子捕获载体pCAMBIA1391Z中,检测RRD3片段是否在植物中也有启动子活性,转基因烟草再生植株和水稻愈伤组织均显示了gusA基因的表达,表明其在单子叶和双子叶植物中均可行使启动子功能。基于RRD3的双效启动子特性,我们设计并构建了植物通用双元RNAi载体pCRiRRD3,适于进行植物的RNA干涉实验研究。我们在植物RNAi载体pCRiRRD3的内含子(200bp)的上下游分别引入了2个多克隆位点,方便了正义及反义片段的接入。转基因烟草植株的GUS组织化学及荧光定量分析表明该载体可有效进行RNA沉默。这些研究结果表明,利用单子叶和双子叶双效活性的RRD3启动子而构建的植物RNAi载体,可同时有效地应用于大部分单子叶和双子叶植物的RNAi实验及研究。
基金
This work was supported by the National Natural Science Foundation of China (No. 30370809).
