【作者】 门潇；

【导师】 杨永华； 卢山；

【作者基本信息】 南京大学 ， 植物学， 2011， 博士

【摘要】 Orange (Or)基因是在天然突变的菜头呈橘黄色的花椰菜中发现的。突变体的菜头、茎髓、叶子基部、顶端分生组织的细胞由于大量积累p-胡萝卜素(能达到野生型的几百倍)而呈现橘黄色。研究发现突变体和野生型类胡萝卜素合成途径(包括上游MEP途径)的酶基因表达水平并没有显著差异：类胡萝卜素含量的上升可能是由于积累能力的提高而不是合成能力的增强。进一步研究发现,野生型的Or基因编码一个质体定位的富含半胱氨酸DnaJ类似蛋白,突变体是由于基因中插入了一个带有长末端重复序列的转座子。Or基因敲除突变体并未表现出色素积累的表型,说明oR.是功能获得性(gain-of-function)突变而不是无义/功能丧失(loss-of-function)突变。or纯合突变体顶端分生组织质体分裂发育受到影响,细胞中仅存在一到两个较大的有色体,但叶片质体发育正常。与有色体形成相关的基因在oR.突变体中表达量升高,且or总是在富含前质体的幼嫩组织里高表达,说明OR与促进有色体的形成有关。最近有研究报道OR与真核翻译释放因子eRF1-2在细胞核里相互作用,认为OR可能是可以在质体-核之间穿梭的双定位蛋白。把oR.基因转土豆、拟南芥等,土豆块茎会积累类胡萝卜素而变黄,拟南芥花序中类胡萝卜素含量也提高,说明OR在基因工程改善作物的营养品质方面具有很好的应用前景。为了进一步了解OR在植物体内的作用机理,本论文中我们以拟南芥or过表达、Or敲除突变体和花椰菜野生型和or.突变体的幼苗为材料,研究了拟南芥突变体与对照组在表型、色素含量、转录组等方面的差异,和花椰菜幼苗去黄化过程中色素含量、质体发育、质体-核信号交流以及相关基因在转录水平、蛋白水平等方面的情况。综合所有的研究结果得到以下结论：1)OR在绿色组织中(如花椰菜子叶、拟南芥整株)也起到了提高类胡萝卜素含量的作用,但是不像在非绿色组织中作用那样明显。与花椰菜or突变体菜头中主要积累的是β-胡萝卜素不同的是,绿色组织中植物黄体素、花药黄素等含量提高了,说明OR的作用具有组织特异性。花椰菜or.突变体幼苗子叶中叶绿素含量没有明显变化,花青素含量明显升高。2)芯片分析的结果表明拟南芥过表达or突变体中类胡萝卜素合成途径的相关基因表达模式并没有显著变化。而定量PCR分析花椰菜野生型和or突变体去黄化幼苗子叶中相关基因的表达发现,突变体类胡萝卜素合成途径上PSY、LCYE、VDE等基因的表达有所上调,PDS的蛋白水平在两者间也不同,表明合成能力提高是突变体幼苗子叶中类胡萝卜素含量略高的原因。因为研究的组织和发育阶段不同,这与之前认为OR的作用是使积累能力增强而不是合成能力提高的结论并不矛盾。3)OR对去黄化过程中质体的发育没有显著影响,除了在6h突变体子叶样品中观察到类囊体比野生型的更厚、垛叠排列松散,这可能与突变体中存在胁迫环境有关。4)花椰菜or.突变体去黄化幼苗子叶中花药黄素和花青素含量升高、VDE表达上升、LHCB和LHCA蛋白水平降低、Fv/Fm下降、NPQ升高、CAT酶活增强、ABA含量高等都说明突变体中存在胁迫环境。芯片的结果也支持了这一点：拟南芥过表达or突变体的花苞和茎生叶中,表达共同上调的基因在一些胁迫条件下或是激素合成、信号传导、抗逆相关突变体中共同上调或下调,暗示着OR在植物调节生理活动、应对环境刺激中起作用。5)花椰菜or突变体中的胁迫环境触发了GUN1的高表达,但是OR并不作用于质体对核信号的四吡咯代谢途径或质体基因表达途径,而是与质体的氧化还原状态和活性氧信号途径有关。突变体菜头中谷胱甘肽总量和GSH/GSSG比值的下降支持了这一点,且突变体中相对氧化的环境有可能造成一些酶(包括类胡萝卜素合成途径的酶)活性增强。6)拟南芥or突变体变得对光尤其是蓝光非常敏感,且OR很有可能联合GUN1和CRY1参与到整合光信号和质体信号的途径中。本研究综合分析了拟南芥和花椰菜两种植物不同发育阶段、不同组织中OR在各个层面的影响,为进一步揭示OR的作用机制提供了可能的研究方向。更多还原

【Abstract】 Orange (Or) gene was discovered in spontaneous, semidominant mutant of cauliflower which showed orange color of its curd, shoot pith, leaf base and shoot apical meristem due to the abnormal accumulation of high level of β-carotene. The homozygous mutant (or) causes no significant up-regulation of genes required for β-carotene biosynthesis or the upstream MEP pathways. It appears that the abnormal accumulation of high level of β-carotene is due to an enhanced ability of accumulation rather than increased capacity of biosynthesis. Further studies revealed that Or encodes a Cys-rich domain containing DnaJ-like protein. The or gene mutation is due to the insertion of a long terminal repeat retrotransposon in the or allele. Or knock-out mutant didn’t show the high pigment accumulation pattern, indicating that the or mutant is not a loss-of-function mutation but a gain-of-function mutation. Plastid division is affected in the shoot apical meristem of or homozygous mutant, and only one or two large plastids were observed in a cell. Plastid development in leaves is normal. Genes involved in chromoplast differentiation are up-regulated in or mutant and or gene itself has a high expression in proplastid-rich or other noncolored plastid-rich tissues, suggesting that the function of OR is associated with a cellular process that promotes the formation of chromoplasts. Recently, OR was reported to interact with eRF1-2in the nucleus of Arabidopsis protoplast and was thought to be a plastid-nucleus bi-localized protein. or transgenic potato and Arabidopsis also showed increased carotenoids content in their tuber and inflorescence respectively, indicating the great potential of or gene in improving nutritional value of crops.To further understand the possible mechanism of OR, we generated or over-expression and Or dsRNA interference Arabidopsis mutants and investigated the phenotype, pigments content, transcriptome profile of the mutants. We also utilized de-etiolated wild-type (WT) and or mutant cauliflower seedlings as research objects. Pigment contents, plastid development, plastid-to-nucleus retrograde signaling pathways, transcription level and protein level of genes involved in the de-etiolation process were analyzed. Taken together, we preliminarily proposed the following conclusions:1) OR also functions in green tissues, such as Arabidopsis leaves and cauliflower cotyledons, although not that effective as in non-green tissues. In contrast to the great enhanced level of β-carotene content in mutant cauliflower curds,the increase of β-carotene content was not significant in green tissues and the contents of lutein and antheraxanthin were also increased, indicating that OR may play its role in a tissue-specific manner. There was no significant change in the content of chlorophylls, while anthocyanin content increased significantly in mutant cauliflower seedlings.2) Microarray analysis of the Arabidopsis mutants showed no remarkable change of the transcription pattern of the genes required for carotenoids biosynthesis. But the Q-PCR results exhibit increased expression level of PSY, LCYE, VDE in de-etiolated or mutant cauliflower cotyledons. The protein levels of PDS were also different between WT and mutant cauliflowers. These might account for the higher content of carotenoids in mutant cauliflower cotyledons. Due to the different tissues and different development stages investigated, the conclusion was not in contradiction with the previous studies.3) OR has no significant effect on the plastid development in de-etiolated cauliflower cotyledons, except for the slightly looser and thicker grana in6h de-etiolated mutant cauliflower cotyledons. This might related to the stress condition in the mutant.4) There were many cues implying that a stress condition existed in the mutant cauliflower seedlings, such as the increased content of antheraxanthin, anthocycanin and ABA, the enhanced expression of VDE, decreased level of Fv/Fm and increased level of NPQ. Microarray analysis also supported the point. The up-regulated genes in both flower buds and cauline leaves of Arabidopsis or mutants were simultaneously up-regulated or down-regulated in stress condition treated WT plants or in resistant-defective mutants, proving that OR may be involved in the regulation of plant physical activity responding to the environmental stimuli.5) The stress condition in homozygous cauliflower or mutant triggered the high expression of GUN1. It appears that or may function in the Redox/ROS pathways rather than tetrapyrroles metabolism pathway or the plastid gene expression pathway. The decreased level of total glutathione and the ratio of GSH/GSSG in or mutant cauliflower curd also supported the point. The more oxidized environment in mutant cauliflower seedlings may increase the activity of the enzymes required for carotenoids biosynthesis.6) or mutant is sensitive to light, especially the blue light. Its close co-expression relationship with CRY1and the high expression level of GUN1in or mutant imply that OR may participate in the integration network of light and plastid signals together with CRY1and GUN1.In this work, comprehensive analyses were carried out at various aspects in Arabidopsis and cauliflower mutants. The conclusions provide a probably orientation for the future work.更多还原