【作者】 郝华玲；

【导师】 毕玉蓉；

【作者基本信息】 兰州大学 ， 植物学， 2013， 硕士

【摘要】 花青素和叶绿素是高等植物中存在的两大类非常重要的色素。花青素存在于植物表皮细胞的液泡中,使高等植物的花、果实、种皮呈现出红、蓝、紫的颜色,花青素在植物中行使着多种重要的生理功能,例如吸引授粉者和种子传播者,保护植物免受昆虫攻击、缓解UV-B辐射以及具有很强的抗氧化活性等。近年的研究表明,花青素的摄入还能够降低人类心血管疾病、糖尿病、关节炎和癌症的发病率；而叶绿素则是高等植物中最为重要的一类色素,它不仅是绿色植物的主要色素,更是植物进行光合作用的重要物质,同时叶绿素在人类的日常生活中也占有非常重要的作用,具有造血、解毒、降低胆固醇以及抗突变的保健功效。因此,对于花青素以及叶绿素的合成代谢及其调控机制的深入研究,对于人类的保健以及增加作物产量都具有非常重要的意义。细胞分裂素作为一类重要的植物激素在植物生长发育的整个过程中起到了非常重要的作用,包括调节植物细胞的分裂以及新陈代谢,诱导叶绿体的发育,调节植物地上部分以及根系的发育,延缓叶片衰老等。本文以模式植物拟南芥为材料,采用生理分析和分子生物学方法研究了细胞分裂素对植物花青素和叶绿素含量的影响,以及红光受体PHYB及光敏色素相互作用因子(Phytochrome-interacting factors, PIFs)在此过程中的调控作用。研究结果表明：(1)光下外源细胞分裂素(6-BA)可诱导拟南芥幼苗中花青素的大量积累,花青素含量随着处理浓度及处理时间的增加而增加,并且花青素合成通路中的三个重要酶,查尔酮合酶(CHS)、查尔酮异构酶(CHI)及二氢黄酮醇-4-还原酶(DFR)的基因表达量明显受到了外源细胞分裂素的上调,说明外源6-BA通过诱导花青素合成基因表达量的增加,从而促进花青素的积累；(2)光下外源6-BA可以显著降低拟南芥幼苗新生叶片中叶绿素的含量,且这种降低没有浓度依赖性,0.1“M6-BA处理效果最为明显；(3)拟南芥中存在三种细胞分裂素受体：AHK2、AHK3和AHK4/CRE1,光下6-BA对拟南芥幼苗花青素以及叶绿素含量的影响主要是通过其受体AHK3与CRE1实现的：(4)6-BA对拟南芥幼苗花青素以及叶绿素含量的影响依赖于光照,并且受红光信号的调控。光下外源6-BA处理下,红光受体PHYB突变体phyB幼苗中花青素积累量明显减少,并且新生叶片中叶绿素的含量变化也较小,phyB中花青素合成基因的表达量明显较野生型低,说明PHYB信号参与调节了光下6-BA对拟南芥幼苗花青素以及叶绿素含量的影响；(5)本研究证明PIFs家族中的PIF4在光下6-BA诱导的拟南芥幼苗花青素及叶绿素含量变化中起到了负调控的作用。光下外源6-BA处理下,对照Col-0、突变体pifl、pif3、pif4、pif5及其过表达系PIF3OX和PIF5OX幼苗中均积累了大量的花青素,并且新生叶片中叶绿素含量也显著下降,但是过表达系PIF4OX中花青素积累量明显较对照少,并且新生叶片中叶绿素的含量变化也较小。另外,PIF4OX幼苗中花青素合成基因的表达量明显较Col-0、 PIF3OK及PIF5OX (?)(?),进一步证明PIF4在6-BA诱导的拟南芥幼苗花青素及叶绿素含量变化中起到的负调控作用。更多还原

【Abstract】 Anthocyanins and chlorophyll are two classes of very important pigments in higher plants. Anthocyanins are the pigments in vacuole of epidermal cell. They have caused the red, blue or purple colour in flowers, fruits and seed coats. Anthocyanins are provided with various important physiological functions in plants. For example, it appeals to pollinators and seed dispersers, defends protects plant against insect attracting and relieves the damage of UV-B radiation, as well as acts as powerful antioxidants etc. Recent studies show that anthocyanins could reduce the incidence of human cardiovascular disease, diabetes, arthritis and cancer. Chlorophyll is an extremely important kind of pigment in higher plants. It is not only an important pigment, but also the critical substance in photosynthesis of plant. Meanwhile, chlorophyll also occupies a very important role in human daily life. It is provided with good effects on hemopoiesis, detoxification, reduce cholesterol level and anti-mutation. Therefore, the further study on the synthesis metabolism of anthocyanins and chlorophyll and its regulation mechanism is essential to human health and increase in crop production. Cytokinins as a kind of phytohormones play critical roles in plant growth and development, including regulation of cell division and metabolism, stimulation chloroplast development, modulation of shoot and root development, and delay of leaf senescence in plants etc. In the present study, the model plant Arabidopsis thaliana was used as material, the effects of cytokinins on anthocyanin and chlorophyll content, and the regulatory role of red light receptor PHYB and PIFs (Phytochrome-interacting factors) in this process were investigated by physiological and molecular biology method. The results showed that:(1) In light, the marked accumulation of anthocyanins Arabidopsis thaliana seedlings was induce by treatment with exogenous cytokinin (6-benzyladenine,6-BA). Levels of mRNAs for three genes of the anthocyanin biosynthesis pathway, chalcone synthase (CHS), chalcone isomerase (CHI), and dihydroflavonol reductase (DFR) were obviously up-regulated by exogenous6-BA. These data suggested that exogenous6-BA promoted anthocyanins accumulation by inducing the expression of anthocyanin synthesis genes.(2) Chlorophyll content in newborn leaves of Arabidopsis thaliana was significantly decreased by6-BA in light. The decrease of chlorophyll content was not concentration-dependent manner. The treatment of0.1μM6-BA showed the maximum efficiency.(3) In Arabidopsis thaliana, there is three cytokinin receptors, AHK2(Arabidopsis his kinase2), AHK3and AHK4/CRE1. We found that AHK3and CRE1played an important role in the changes of anthocyanin and chlorophyll content induced by6-BA in light.(4) Effects of6-BA on anthocyanin and chlorophyll content were dependent on light and regulated by red light signal. The anthocyanin accumulation content in red light photoreceptor PHYB mutant seedlings was marked decrease and the change of chlorophyll content in newborn leaves of it was also less than that of the control in the exogenous6-BA treatment under light. The expression levels of three genes of anthocyanin biosynthesis pathway in phyB were much less than that of control (Col-0) in the exogenous6-BA treatment under light. These results indicated that PHYB signal was involved in regulating the effects of cytokinins on the changes of anthocyanin and chlorophyll content in Arabidopsis seedlings under light.(5) Our data indicated that PIF4negatively regulated the changes of anthocyanin and chlorophyll contents induced by6-BA. In6-BA treatments, the anthocyanin in control (Col-0), mutants (pif1, pif3, pif4, pif5) and over-expression line (PIF3OX and PIF5OX) seedlings obviously accumulated, and the chlorophyll contents in the newborn leaves of them were also marked decrease. But the anthocyanin accumulation content in PIF4OX seedlings was much smaller than that of in control in6-BA treatment. So was in changes of cholorphyll content in newborn leaves of it. Others, the expression levels of anthocyanin biosynthesis genes in PIF3OX and PIF5OX seedlings were much less than that of Col-0, PIF3OX and PIF5OX in6-BA treatment. The present data indicated that PIF4in Arabidopsis seedlings was the essential role in the changes of anthocyanin and chlorophyll contents induced by6-BA.更多还原