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在植物抗病基因工程中,目标基因可控的高效表达是一重要目标。在前期的研究中,我们将串联的病原物高度特异性诱导启动子EAS4(EP)和hsr203J(HP)转入烟草,uidA基因在相应病原物或激发子诱导后可被驱动表达。为进一步研究此串联双启动子驱动的基因表达特性,采用荧光法对GUS诱导表达活性进行了定量检测。结果发现,双启动子表现较高的诱导表达活性,双启动子与单启动子的活性差异均达极显著水平。研究还发现,两个启动子串联的顺序对其活性强度和时间动态有影响,双启动子HP+EP的诱导表达高峰出现在诱导后6～10 h,而EP+HP的活性高峰出现在诱导后8～14 h。同时,初步分析了串联的EP和HP协同促进基因高水平表达的原因。串联的双病原物特异性诱导启动子不但可响应较广谱的病原物的诱导表达,而且可协同促进基因的高效表达,因而在植物抗病基因工程中具有广阔的应用前景。
In plant disease resistance gene engineering, the controlled expression of the target gene is an important goal. In the previous study, we transferred the highly pathogen-specific promoters EAS4 (EP) and hsr203J (HP) into tobacco in series, and the uidA gene can be driven to express after induced by the corresponding pathogen or elicitor. To further investigate the gene expression characteristics driven by this tandem double promoter, the GUS-induced expression activity was quantitatively detected by fluorescence method. The results showed that the double promoter showed higher induction activity, the difference between the two promoters and the single promoter activity reached extremely significant level. The study also found that the order of the two promoters had an impact on the activity intensity and time dynamics. The peak expression of the dual promoter HP + EP occurred 6 to 10 h after induction, while the peak of the activity of EP + HP appeared in the induction After 8 ~ 14 h. At the same time, the reason why tandem EP and HP cooperate to promote high level expression of genes was analyzed. The tandem two-pathogen-specific inducible promoter not only can respond to the induced expression of a broader spectrum of pathogens, but also can synergistically promote the high expression of the gene, thus having broad application prospect in plant disease resistance gene engineering.