串联双病原物诱导启动子驱动基因表达的特性

 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。

Characteristics of gene expression driven by two tandem pathogen-inducible promoters

High-level expression of exogenous genes under controlled conditions is an important target of genetic engineering for plant disease resistance. From our previous research it was showed that in response to induction of certain pathogens or elicitor, uidA in transgenic tobacco plants could be driven to express under the control of two highly pathogen-inducible promoters, concatenated by EAS4 and hsr203J promoter. In order to further explore the characteristics of gene expression under the control of the tandem promoters, the induced GUS activity was quantitatively assayed by the fluorometric reaction method of Jefferson. It was found that the tandem promoters had higher induced activity, and the activity of tandem promoters is very different from that of single promoter. Furthermore, the tandem order of the two promoters had also an effect on intensity and time course of induced GUS activity. Dynamic assay of induced GUS activity showed that the tandem HP+EP promoters reached the highest value in expression activity after 6-10 hour of induction, while the highest value occurred in the tandem EP+HP promoters after 8-14 hours. In the end, the causes are discussed for cooperatively elevating the expression level of uidA by tandem EP and HP promoters. Two tandem pathogen-inducible promoters can not only make transgenic plants responsive to more pathogens but also boost expression level of exogenous genes, will therefore be widely applied in genetic engineering of plants for disease resis-tance.