【作者】 王曼；

【导师】 王小菁；

【作者基本信息】 华南师范大学 ， 植物学， 2003， 博士

【摘要】 高等植物花色素苷积累和CHS基因表达受光调控，蓝光是最有效光谱之一。尽管对蓝光诱导的反应机理和信号转导途径已有一些研究，但对胞外钙离子和钙调素（CaM）是否参与反应仍不清楚。本文以拟南芥（Arabidopsis thaliana，Lansberg生态型）的野生型（WT）和hy4突变体为材料，在研究蓝光诱导花色素苷积累和CHS基因表达的基础上，对参与蓝光反应的信号转导途径组分进行了研究，主要结果如下： 1 蓝光诱导拟南芥花色素苷积累和CHS基因表达 在20 μ mol·m^-2·s^-1白光下生长13d龄的幼苗，经不同光强和不同时间的蓝光处理后，叶片花色素苷积累显著增加。蓝光诱导花色素苷积累的作用具有对光强的依赖性，随着光强的升高，其花色素苷的含量增加；同一光强下照光时间延长，含量增加。蓝光对拟南芥叶片花色素苷积累的诱导是高辐照反应。蓝光处理后，hy4叶片的花色素苷含量明显低于WT中的花色素苷含量，说明cry1是蓝光诱导花色素苷积累的主要光受体。 蓝光诱导拟南芥WT中CHS基因的表达，蓝光处理4小时即有表达，8小时达到最高的表达，之后表达量逐渐下降；在突变体hy4中，蓝光诱导CHS基因几乎不表达。说明cry1介导蓝光诱导CHS基因的表达。 2 蓝光诱导花色素苷积累和CHS表达的信号转导组分研究 1)Ca²⁺和CaM的作用 蓝光诱导叶片花色素苷的积累需要培养基中Ca²⁺的存在，无Ca²⁺条件下蓝光反应受阻；培养基中加入Ca²⁺载体A23187，促进蓝光诱导的花色素苷积累；EGTA和质膜Ca²⁺通道抑制剂异博定（verapamil）、尼群地平（nifedipine，Nif）处理则抑制蓝光诱导的花色素苷积累。 A23187对蓝光诱导的CHS基因表达有一定的促进作用，EGTA和异博定则有抑制作用；尼群地平（Nif）对花色素着积累和基因表达的作用不一致，需要进一步研究。上述实验表明蓝光反应需要胞外C／”的参与。 IP3与 CS＞通道受体结合抑制剂肝素处理可促进蓝光诱导的花色素苦积累，但抑制CHS基因表达；IP3分解抑制剂LIC13则抑制花色素着积累而促进基因表达；施加肌醇既促进蓝光诱导花色素着积累又促进基因的表达。看来，IP3敏感的CaZ”通道对蓝光诱导的CHS基因表达起正调节的作用。 钙调素KaM)桔抗剂Tfo和W7对花色素昔的积累和CHS基因表达的作用有抑制也有促进。 lP3敏感的胞内钙库 CaZ”通道和 CaM是否参与 cry介导的特异蓝光反应的信号转导还需深入研究。蓝光诱导的花色素音积累和CHS基因表达可能存在着精细的CaZ”／CaM信号途径。 对冲 突变体的研究表明，大部分影响蓝光诱导WT花色素苦积累和CHS表达的o’”相关药物女 A23 87、EGm、Nif、veraplllllll、肝素、肌醇等处理到对你4没有明显影响，说明CaU参与的蓝光反应是由cryl介导的特异蓝光反应。2)质膜组分的作用 运用黄素蛋白括抗剂DPI进行的实验表明，质膜氧化还原参与蓝光诱导的拟南芥花色素着积累及 M基因表达的信号转导过程，这一过程是 cry介导的；质膜H”一ATPase和 C／一ATPase在蓝光诱导的 CHS表达及花色素着积累的信号途径中起着负调节的作用；H”一 ATPase的作用与 Cyl介导的特异蓝光反应有关。 G蛋白抑制剂PTX促进蓝光诱导的花色素苦积累，G蛋白激活剂CTX有相反的作用；但二、者都抑制 CHS基因的表达。G蛋白的作用并非与 Cry介导的特异蓝光反应相关。3)其它组分的作用 蛋白激酶抑制剂星形抱菌素（staurosporine）和磷酸酶抑制剂斑鳌素＊antheddin)的实验表明，staurosporine敏感的蛋白激酶是蓝光诱导的花色素苦积累和基因表达的负调节因子；而cantheddin敏感的磷酸酶是蓝光诱导CHS表达的正调节团于。蛋白质的可逆磷酸化可能参与了 Cry介导的特异蓝光反应。 蛋白质合成抑制剂放线菌酮＊，Chx)明显抑制蓝光诱导的花色素着积累和CHS的表达，合成新的蛋白质是Cry回介导的特异蓝光反应所必需的。4)糖的作用 糖不仅作为碳源参与花色素音的积累，还可能作为信号分子参与蓝光诱导的CHS表达。更多还原

【Abstract】 Numerous experiments have indicated that anthocyanin biosynthesis in higher plants is regulated by light and blue light is one of the most effective factors to stimulate anthocyanin accumulation in seedlings. Although several reports have been published for the components involved in signaling pathway of light-controlled pigmentation, evidence of extracellular Ca2+and CaM involved in blue light induced anthocyanin accumulation and CHS expression is still limited.WT and hy4 of Arabidopsis thaliana (Landsberg) were used in the experiments. The components involved in the signaling pathway of blue-light-induced anthocyanin accumulation and CHS expression were investigated by pharmacological approach. The main results are as follows:1. Blue-light-induced anthocyanin accumulation and CHS expression inArabidopsis13-d-old seedlings of WT and hy4 Arabidopsis grown under white light (WL) with the light fluence rate of 20μmol·m-2·s-1 were transferred into blue light (BL) and irradiated for period of time with different fluence rates before harvesting. The leaves isolated from the seedlings showed an increase in the anthocyanin content. The BL response was fluence dependent and the anthocyanin content increased with the irradiating time under the same fluence rate. Our results supplied another evidence that blue-light-induced anthocyanin accumulation was a high irradiance response (HIR) in plant photomorphogenesis. Anthocyanin content in hy4 mutant was lower than that in WT Arabidopsis after BL treatment and it was demonstrated that cryptochrome 1 (cry1) mediated the BL response in anthocyanin accumulation.Chalcone synthase (CHS) gene expression in the WT leaves was induced by BL and it could be detected at 4 h and reached a peak at 8 h in BL. And then the gene expression declined. Blue light could not induce any gene expression in hy4 mutant indicating cryl is the photoreceptor mediating the blue-light-induced CHS gene expression.2. Components involved in blue-light-induced anthocyanin accumulation andCHS expression in Arabidopsis1) Ca2+/ CaMThe Ca2+ in the medium was essential for the blue-light-induced anthocyanin accumulation. BL response was blocked without Ca2+ and could be enhanced by the application of A23187, a calcium ionophore. The anthocyanin accumulation induced by BL was inhibited by EGTA and two nonpermeable L-type channel blocker verapamil and nifedipine (Nif). CHS gene expression induced by BL was enhanced by A23187 and inhibited by EGTA and verapamil. But the effect of another Ca2+ channel blocker Nif on the gene expression was opposite to its role in the inhibition of anthocyanin accumulation. Our data demonstrated that extracellular Ca2+ was required for blue-light-induced anthocyanin accumulation and CHS gene expression and verapamil sensitive plasma membrane Ca2+ channel played a positive effector on BL response.Heparin, an antagonist of IP3 receptor increased the anthocyanin content induced by BL but inhibited CHS gene expression. LiCl3, an inhibitor of IP3 pathway, showed an inhibition of the pigmentation but had a positive effect on gene expression. Inositol supplied in the medium enhanced both blue-light-induced pigmentation and gene expression.Antagonists of CaM trifluoperazine (Tfp) and W7 were used for detecting the role of CaM in BL response. It was showed that the former had a positive and the later had a negative effect on BL response.There is still a question to be addressed whether intracellular IP3 sensitive calcium store and CaM are involved in BL response mediated by cry 1. There should be a fine regulating process of Ca2+/CaM in the blue-light-induced anthocyanin accumulation and CHS gene expression in Arabidopsis.Application of most pharmacological chemicals such as A23187, EGTA, Nif, verapamil, heparin and inositol did not show significant effect on athocyanin accumulation after BL treatment in hy4 mutant. This indicated that the Ca2+ play an important role in cryl specific signaling.2) Components in plasma membraneE更多还原