Molecular analysis of GLP(germin-like protein)genes related to photoperiodic response.

为了阐明光周期诱导开花的分子基础，我们研究了短日照诱导的牵牛子叶中基因表达的差异。我们鉴定了一个基因，其转录物在花诱导黑暗期积累在叶和子叶中，并命名为PnGLP（P.nil germin-like protein）。PnGLP是Germin和GLP基因的叶型亚家族的成员。北方分析表明，PnGLP mRNA的水平表现出昼夜节律，在ZT 10左右（黑暗期开始后10 h）达到最大值，并且这种振荡在延长的连续黑暗中继续。这种表达模式显示了对CAB（叶绿素a/b结合蛋白）的反相，CAB是研究最广泛的基因。为了研究叶型GLP基因调控与光周期性的关系，分离并鉴定了拟南芥（Arabidopsis）中PnGLP的同源物AtGLP 1和AtGLP 2。AtGLP 1和AtGLP 2 mRNA在叶片中的积累也表现出昼夜节律，在ZT 14左右（光照开始后14 h）达到最大值。这种振荡主要在连续光照下持续。因此，AtGLP 1和AtGLP 2的调控模式与PnGLP相反，可能反映了短日植物和长日植物的生理差异。我们分析了这些基因的启动子区的核苷酸序列测定和功能分析，使用荧光素酶基因作为报告。在这三种启动子中均未发现保守的顺式元件，但均观察到几个加塔元件。在AtGLP 2启动子中，我们发现了两个可能与连续光照下昼夜节律表达有关的加塔元件。我们推测有几种GATA结合蛋白在昼夜节律的特定时间控制时钟控制基因的表达。我们的实验目前正在进行这种假设。

To clarify the molecular basis of the photoperiodic induction of flowering, we studied the differences in gene expression in short-day induced cotyledons of Pharbitis nil cv.Violet. We identified a gene whose transcript is accumulated in leaves and cotyledons during the flower-inductive dark period, and designated PnGLP(P.nil germin-like protein). PnGLP is a member of the leaf-type subfamily of Germin and GLP genes. Northern analysis revealed that the level of PnGLP mRNA showed a circadian rhythm, with maxima around ZT10(10 h after the beginning of the dark period), and this oscillation continued in lengthened continuous darkness. This pattern of expression showed an antiphase against CAB(chlorophyll a/b-binding protein) that is of the most widely studied gene. To study the molecular mechanisms of leaf-type GLP gene regulation in relation to photoperiodism, homologues of PnGLP in Arabidopsis(a long-day plant), namely, AtGLP1 and AtGLP2 were isolated and characterized. Accumulation of AtGLP1 and AtGLP2 mRNA in leaf also showed circadian rhythms, with maxima around ZT14(14 h after the beginning of the light period). This oscillation continued mostly under continuous light. Thus, the mode of regulation in AtGLP1 and AtGLP2 showed opposite to that of PnGLP, possibly reflecting the physiological differences between short-day plants and long-day plants. We analyzed promoter region of these genes by means of determination of nucleotide sequence and functional analysis using a luciferase gene as a reporter. We did not find any conserved cis-elements among there promoters, but observed several GATA elements in all the three promoters. In case of AtGLP2 promoter, we found two GATA elements that may responsible to the circadian expression under the continuous light. We speculated that there are several GATA-binding proteins that govern expression of clock-controlled genes at specific timings during circadian time. Our experiment is currently underway on such hypothesis.