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堇菜（Pharbitis nil cv.Violet）子叶中基因表达的差异。我们发现了一个在花诱导暗期在叶片和子叶中积累转录的基因，并将其命名为pglp (P。无发芽样蛋白)。pingglp是Germin和GLP基因叶型亚家族的成员。Northern分析显示，pglp mRNA水平具有昼夜节律性，在ZT10左右（暗期开始后10 h）达到峰值，这种振荡在延长的连续黑暗中持续。这种表达模式显示了对CAB（叶绿素a/b结合蛋白）的反相，这是研究最广泛的基因。为了研究叶片型GLP基因与光周期发育相关的调控分子机制，我们在拟南芥（长日照植物）中分离并鉴定了pglp的同源基因AtGLP1和AtGLP2。叶片中AtGLP1和AtGLP2 mRNA的积累也具有昼夜节律，在ZT14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继续。因此，AtGLP1和AtGLP2的调控模式与pglp相反，可能反映了短日照植物与长日照植物的生理差异。我们通过核苷酸序列测定和荧光素酶基因作为报告基因的功能分析来分析这些基因的启动子区域。我们在这三个启动子中没有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一些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昼夜节律表达有关的GATA元件。我们推测有几种gata结合蛋白在昼夜节律时间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中。

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.