【作者】 高慧君；

【导师】 徐娟； 邓秀新；

【作者基本信息】 华中农业大学 ， 果树学， 2013， 博士

【摘要】 类胡萝卜素是自然界中常见的物质,是植物生存必需的次生代谢物。它参与众多生物学过程,对人类健康及动植物生存具有重要意义,它赋予花卉果实丰富色彩,有利于授粉和种子传播。因此,深入理解植物中类胡萝卜素的代谢调控机制有助于我们更好地利用类胡萝卜素,使其服务于我们的生活。柑橘愈伤组织本身含有多种类胡萝卜素,又因其生长周期短、培养条件简易可控等特点,使之成为植物类胡萝卜素代谢研究的较理想材料。本研究通过多种外界因素处理柑橘愈伤组织,包括光暗、除草剂、生长周期、糖分和激素处理,深入分析了不同因素对柑橘愈伤组织中类胡萝卜素代谢调控特征及类胡萝卜素代谢同其它生物学过程的关系。主要研究结果如下：1、对红马叙葡萄柚(Red Marsh grapefruit)(Citrus paradisi Macf.)、默科特橘橙(Murcott tangor)(C.reticulata×C. sinenis)、塔罗科血橙(Tarocco blood orange)(C.sinensis[L.] Osbeck)和冰糖橙甜橙(Bingtangcheng sweet orange)(C.sinensis[L.] Osbeck)4种柑橘胚性愈伤组织进行光照/黑暗处理。HPLC和Real-Time PCR分析表明,光照对柑橘愈伤组织中类胡萝卜素的调控模式因受基因型影响呈现多样性。在光照条件下,红马叙葡萄柚愈伤组织中类胡萝卜素的合成受阻；而塔罗科血橙愈伤组织中类胡萝卜素受光照诱导积累。环氧类胡萝卜素的光稳定性强于非环氧类胡萝卜素,如花药黄质和紫黄质。同时本实验发现PSY是2种甜橙愈伤组织中类胡萝卜素合的限速基因；而在默科特橘橙愈伤组织中,光照强烈诱导CRTISO的表达。2、CPTA是胡萝卜素环化酶抑制剂,用0.2%(w/v)浓度处理红马叙愈伤组织,结果表明CPTA不仅能有效地促进类胡萝卜素(特别是番茄红素)的合成积累,还能刺激一些次生代谢物的合成,例如类黄酮和生物碱类物质,但对初生代谢物的生物合成影响不大。利用基因芯片分析处理前后愈伤组织中转录水平上的差异,其中33个基因上调,90个基因下调。这些基因包括编码过氧化物酶、抗逆相关基因和P450等。同时,检测到过氧化氢(H2O2)、JA、SA和ABA含量下降。本研究认为,红马叙愈伤组织在CPTA的刺激下产生大量抗氧化性物质(特别是类胡萝卜素和类黄酮)并可能通过非酶促反应缓解了细胞内的氧化胁迫,避免了激活由激素介导的抗逆机制。同时,本研究发现CPTA处理后IAA的增加可能是引起类胡萝卜素总量激增的部分原因。3、利用基因芯片对自然生长状态下(10DAC和30DAC)的愈伤组织进行分析。结果表明,共有2505个差异表达基因,其中上调1154个,下调1351个,并利用Real-Time PCR的方法对基因芯片结果进行验证。初生代谢组分析表明大部分可溶性糖和有机酸的含量显著下降,而氨基酸和脂类含量变化不大,这暗示愈伤组织在生长后期缺乏碳水化合物提供能量。结合芯片数据分析发现,糖分的匮乏可能引起TCA循环的加速以弥补能量供给不足,同时可能与抗氰呼吸清除细胞内自由基有关;而碳链在逆境环境下有向次生代谢物流动的趋势。在生长后期,伴随类胡萝卜素含量急剧的减少,类胡萝卜素合成相关基因显著上调,说明类胡萝卜素含量的降低可能是多因素导致。H202含量的增加也表明愈伤组织受到氧化胁迫。本研究认为,生长后期糖匮乏可能引起了愈伤组织中H202含量的增加,而H202对类胡萝卜素的积累有一定影响,同时糖分和激素对类胡萝卜素合成的调控有待深入研究。4、在培养基中添加不同浓度蔗糖或葡萄糖,以研究糖分对类胡萝卜素调控的作用。结果表明,外源糖分能刺激愈伤组织中类胡萝卜素的合成,但在较高糖分浓度下,类胡萝卜素的合成受到抑制。Real-Time PCR分析表明外源糖分并不能诱导类胡萝卜素合成相关基因表达,60mM葡萄糖甚至会抑制类胡萝卜素合成相关基因表达。但外源糖分能明显诱导糖转运及糖信号基因的表达,如SUT, TMT和HXK,说明糖可能是愈伤组织中类胡萝卜素合成的信号因子。5、使用外源植物激素ABA、IAA、GA和JA分别处理红马叙愈伤组织12h,3d和10d。结果发现,处理3d和10d后类胡萝卜素积累普遍显著性升高,其中以IAA的效果最为显著。随着处理时间的延长,激素对类胡萝卜素的刺激作用略有降低,但仍高于对照。Real-Time PCR分析结果表明,不同激素对类胡萝卜素转录调控模式存在差异。而对激素相关基因的表达分析发现,属于MADS-box转录因子家族的基因AGL6可能与类胡萝卜素合成有关。综上所述,本研究对理解植物类胡萝卜素代谢的生物学本质具有重要价值,对类胡萝卜素调控进行了进一步的探索,为揭示类胡萝卜素调控机理提供了新的和重要的线索。更多还原

【Abstract】 Carotenoids are essential plant secondary metabolites in nature, which are required for the plant survival. They are involved in many biological processes, and essential for human health and animal/plant life. They give rich colors to the flowers and fruits which are conducive to the pollination and seed dispersal. Therefore, to exert their effective usage, it is essential to understand the regulation mechanism of plant carotenoid metabolism.Citrus callus exhibits extensive diversity of carotenoid patterns, its characteristics like short growth cycle and controllable culture conditions making it to be an ideal material for plant carotenoids metabolism study. In this study, upon various external treatments on citrus callus, including light/dark, herbicides, growth cycle, sugar and hormone, in-depth analysis on metabolic regulation of carotenoid metabolism and other related biological processes was possible. The main results are as follows:1. Four citrus callus including Red Marsh grapefruit (Citrus paradisi Macf.), Murcott tangor (C. reticulata×C. sinenis), Tarocco blood orange (C. sinensis Osbeck) and Bingtangcheng sweet orange (C. sinensis L. Osbeck) were under light/dark treatment. HPLC and Real-Time PCR analysis showed that, light has various regulation patterns of carotenoids in citrus callus depending on the genotype diversity. Under the light, carotenoid synthesis in Red Marsh grapefruit callus was blocked, while light induced carotenoid accumulation in Tarocco blood orange callus. Epoxy carotenoids were more photostability than non-epoxy carotenoids, such as antheraxanthin and violaxanthin. At the same time, it also showed the PSY was the rate limiting gene in2sweet orange callus, while light strongly induced CRTISO expression in Murcott tangor callus.2. CPTA is carotene cyclase inhibitor. In metabolic analysis on Red Marsh callus treated with0.2%(w/v) CPTA showed that not only carotenoids synthesis (particularly lycopene) were promoted, but also the synthesis of secondary metabolites such as flavonoids and alkaloids were stimulated. However, little impact on primary metabolite biosynthesis was detected. Gene microarray analysis was performed to investigate global transcriptional alterations, of which33genes were up-regulated and90genes were down-regulated such as those encoding peroxidase, resistance related and P450. On the other hand, the contents of H2O2, JA, SA and ABA were decreased in treated callus. Our result suggests that CPTA not only greatly promoted the accumulation of carotenoids, but also boosted the production of other antioxidants, especially flavonoids. It was speculated that these potent biological antioxidants play an important role in eliminating oxidative stress in callus caused by CPTA, so that the common warning signals system (hormones and peroxidase) had not been activated. Meanwhile, our study indicated IAA might have some impact on carotenoids accumulation.3. Gene microarray analysis was performed on citrus callus of10days after subculture (10DAC) and30DAC without any treatment. The results showed that, there were2505differentially expressed genes, including1154up-regulated,1351down-regulated. Real-Time PCR validation indicated a good consistence between the two methods. The primary metabolite analysis indicated that the contents of soluble sugars and organic acids were most significantly decreased, while there were slight changes in contents of amino acids and lipids, implying the lack of carbohydrates in callus in the later growth stage. Combined with microarray data analysis, we found that sugar shortage might cause the acceleration of TCA cycle to compensate for the energy supplying shortage, and the cyanide-resistant respiration associated with free radicals clearage, while the carbon chains were rechanneled to the secondary metabolize under stress. In the later growth stage, accompanied with a sharp decrease in carotenoid content, carotenogenesis were significantly increased, while the increase of H2O2indicated an oxidative stress in citrus callus. This study suggested that sugar shortage caused the increase of H2O2, which consequently impacted on carotenoids accumulation. The sugar and hormone induced carotenoids accumulation remains to be elucidated.4. To study the regulation of sugar on carotenoids, different concentrations of sucrose or glucose were added in the medium. The results showed that exogenous sugar could stimulate carotenoid synthesis, but the biosynthesis was inhibited at higher sugar concentrations. Real-Time PCR analysis showed that exogenous sugars could induce carotenogenic genes, however, carotenogenesis was inhibited by high glucose level (60mM), exogenous sugars could induce gene expression which encoding sugar transporter and sugar signals, e.g. SUT, TMT and HXK. Sugar might be taken as a signal affecting carotenoid synthesis to some extent.5. Red Mash citrus callus was treated with exogenous plant hormone ABA, IAA, GA and JA for12h,3d and10d, respectively, to investigate their effects on carotenoids biosynthesis. The results showed that carotenoids accumulated significantly after3d and10d’s treatment, in which IAA was the most effective hormone. As treatment time prolonged, stimulation effect exerted by hormone decreased, but the content of carotenoids kept higher than that of the control. Real-Time PCR analysis suggested that, different hormones had different regulation modes on carotenoid biosynthesis at transcription level. Interestingly, hormone treatments and related gene expression analysis indicated that gene AGL6belonging to MADS-box transcription factor family might be associated with the carotenoids synthesis.In summary, this study has important value in understanding the fundamental biology of plant carotenoid metabolism, and had further explored on carotenoid regulation, and provides novel and important clues in revealing the mechanism of the regulation of carotenoids.更多还原