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紫罗兰(Pharbitis nil cv.Violet)子叶基因表达的差异。我们鉴定了一个基因，其转录产物在花诱导暗期间在叶片和子叶中积累，并命名为PnGLP(P.nil Germin-like Protein)。PnGLP是Germin和GLP基因叶型亚家族的成员。Northern分析表明，PnGLP mRNA的表达呈昼夜节律，峰值在ZT10左右(暗期开始后10h)，这种振荡在延长的连续黑暗中持续。这种表达模式显示出与CAB(叶绿素a/b结合蛋白)的反相，CAB是研究最广泛的基因。为了研究与光周期有关的叶型GLP基因调控的分子机制，我们分离并鉴定了拟南芥(一种长日照植物)中PnGLP的同源基因AtGLP1和AtGLP2。叶片中AtGLP1和AtGLP2mRNA的积累也表现出昼夜节律，在ZT14左右(光照开始后14h)达到最大值。这种振荡大部分是在连续光照下持续的。因此，AtGLP1和AtGLP2的调控模式与PnGLP相反，可能反映了短日照植物和长日照植物之间的生理差异。我们以荧光素酶基因为报告基因，通过核苷酸序列测定和功能分析，对这些基因的启动子区域进行了分析。我们没有在三个启动子中发现任何保守的顺式元件，但在所有三个启动子中都观察到了几个GATA元件。在AtGLP2启动子的情况下，我们发现了两个可能与连续光照下的昼夜节律表达有关的GATA元件。我们推测，在昼夜节律的特定时间，有几个GATA结合蛋白控制着时钟控制基因的表达。我们的实验目前正在基于这样的假设进行。

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.