【作者】 曹洪波；

【导师】 邓秀新；

【作者基本信息】 华中农业大学 ， 果树学， 2012， 博士

【摘要】 类胡萝卜素是植物生存必需的次生代谢类物质,参与众多的生物学过程,如光合作用、抗氧化保护、激素合成以及传粉等。类胡萝卜素也是人类健康所必需的生物活性成份,如提供维生素A前体,抗氧化和衰老,降低心血管疾病风险,以及参与人体免疫、胚胎发育和生殖等。植物是自然界中类胡萝卜素合成的主要生物体,也是人体中类胡萝卜素的最主要来源。因此,研究植物类胡萝卜素代谢调控机制是有效利用类胡萝卜素的重要基础。柑橘中含有丰富多样的类胡萝卜素资源,是植物类胡萝卜素代谢研究的极好材料。本研究通过创制多种柑橘材料,包括转基因胚性愈伤和植株,以及夏橙DH系,深入分析了柑橘类胡萝卜素代谢调控特征、类胡萝卜素代谢同其他生物学过程的关系,以及甜橙中两个类胡萝卜素合成关键基因的等位多态性。主要研究结果如下：1.转CrtB和DSM2基因调控柑橘胚性愈伤中类胡萝卜素积累的研究(1)利用CrtB(来源于细菌的八氢番茄红素合成酶基因)转化马叙葡萄柚(Citrus paradise Macf.)、星露比葡萄柚(Citrus paradise Macf.)、红肉脐橙[Citrus sinensis (L.)Osb.]和日辉杂柑[Citrus reticulata Blanco×(Citrus paradisi Macf.×Citrus Reticulate)]4种基因型的柑橘胚性愈伤组织,创制了一批具有多种类胡萝卜素积累特征的细胞系(ECMs, Engineering Cell Models)。表型观察和HPLC分析表明,ECMs中的类胡萝卜素含量显著高于野生型,而类胡萝卜素的积累模式受基因型影响呈现多样性。在ECMs中检测到30种自由态类胡萝卜素,其中包括野生型含有的所有类胡萝卜素和一些新出现的种类,如番茄红素等。(2)与野生型不同,ECMs中的类胡萝卜素积累具有明显的β路径优势,表现为丰富的p-胡萝卜素积累特征。特别是在暗培养条件下,ECMs的p路径优势更为明显。基于所获数据,本研究提出一种模型用于解释ECMs中出现的p路径优势。该模型认为上游类胡萝卜素的合成能力和LCYE(番茄红素ε环化酶)的瓶颈作用是导致p路径优势的主要原因。(3)在光照条件下,ECMs中的β-胡萝卜素和八氢番茄红素含量显著减少,而叶黄素含量变化不显著。37℃条件下,ECM中多数类胡萝卜素的积累量同样表现出显著的降低趋势,但玉米黄质含量明显提高。另外,可能由于表观调控的影响,同一份ECM中存在CrtB基因表达和类胡萝卜素积累的不均一性特征。(4)选取4种同胚性愈伤相同基因型的成熟果实有色层组织作为类胡萝卜素积累的自然参比。本研究首先分析了ECMs同有色层组织间的类胡萝卜素积累差异。结果表明,有色层中主要积累紫黄质和隐黄质,并多以酯化形式存在；或者以积累番茄红素为主。此外,ECMs和有色层细胞的质体发育形式不同,在ECMs中类胡萝卜素主要以晶体形式存储于淀粉体中;而在有色层细胞中,类胡萝卜素存储于有色体。本研究在ECMs液泡中观察到富含类胡萝卜素的聚集体,这一现象暗示可能存在一种新的类胡萝卜素降解途径。(5)转录分析表明,有色层中类胡萝卜素代谢相关基因的转录水平普遍高于愈伤,特别是HYD(β-胡萝卜素羟化酶基因)具有显著的有色层特异性表达特征,而在愈伤中被抑制。进一步通过CrtB和DSM2(来源于水稻的HYD基因)共表达分析证实,HYD是控制两种组织间类胡萝卜素积累差异的主要位点。在共表达体系中,观察到β-胡萝卜素含量同类胡萝卜素晶体大小显著正相关,这一特征表明类胡萝卜素晶体主要由β-胡萝卜素构成。另外,ECMs中存在内源类胡萝卜素代谢相关基因的表达波动,其中在星露比葡萄柚的转CrtB基因愈伤系中最为明显。2.类胡萝卜素积累对质体发育和代谢的影响(1)在转CrtB基因柑橘愈伤系(ECMs)中,淀粉含量显著低于野生型,而可溶性糖含量高于野生型。这些特征表明,类胡萝卜素积累影响了愈伤组织中的碳水化合物代谢,并且暗示ECMs中存在潜在的有色体发育过程。进一步通过糖饥饿处理证明,ECMs中确实存在有色体发育,只是在正常条件下淀粉的优势积累抑制了ECMs中有色体的形成。(2)本研究利用柑橘芯片分析了4组ECMs同野生型间的转录谱差异。结果表明,4组材料间存在类似的差异基因响应模式。其中最主要的特点是,大量逆境和氧化还原响应相关基因在ECMs中上调表达,如POD(过氧化物酶基因)、GST(谷胱甘肽S转移酶基因)、HSP(热激蛋白基因)和苯丙烷代谢相关基因等。另外,本研究检测到花青素代谢相关基因在ECMs中呈现下调表达趋势,这一特征暗示类胡萝卜素积累负调控花青素合成。在ECMs中,检测到一个直接参与淀粉降解的α-淀粉酶基因呈上调表达趋势,它可能是影响柑橘愈伤组织中淀粉含量变化的重要原因。(3)ABA含量调查表明其不是导致ECMs中逆境基因响应的主要因子。进一步的活性氧(ROS)分析显示,类胡萝卜素积累改变了愈伤组织中的氧化还原状态。主要表现为,ECMs细胞中02-(超氧阴离子)的含量显著降低,而H202(过氧化氢)含量升高。高浓度蔗糖以及还原剂处理ECM的表型特征暗示,ECMs中可能存在02-参与的类胡萝卜素降解反应。另外,本研究认为,细胞氧化还原状态的改变可能是影响柑橘愈伤组织中淀粉含量变化的另一因素。(4)为了证实芯片数据所暗示的类胡萝卜素积累对花青素合成的负调控关系,本研究利用CrtB基因转化具有花青素积累特征的苹果愈伤组织,创制了一份苹果ECM系。该ECM系中的类胡萝卜素含量显著提高,而花青素积累明显受到抑制。用植物类胡萝卜素合成抑制剂(Norflurazon)处理ECM,可以部分恢复ECM中的花青素积累的表型。进一步的转录分析表明,花青素合成基因在ECM中下调表达。3.转CrtB基因早实枳和山金柑的再生及其类胡萝卜素积累特征(1)利用CrtB基因转化柑橘早花资源,获得1株早实枳[Poncirus trifoliate (L.) Raf]和2株山金柑(Fortunella hindsii Swingle)转基因系。由于类胡萝卜素的积累,转基因早实枳的幼芽表现明显的橙色,但多数橙色芽由于不能转绿而最终停止生长。一株转绿后的再生芽被培养成植株,然而它表现出明显的矮化特征。(2)转基因山金柑的绿色组织不表现高类胡萝卜素积累特征,但其花瓣、衰老叶柄、种子以及播种实生苗的根系均表现出橙色表型,显示了明显的类胡萝卜素积累。野生型播种实生苗的根系在光照条件下可以转绿,而转基因实生苗的根系保持橙色表型。另外,转基因山金柑TSJ-1系具有两种色泽的果实,一种表现为同野生型类似的橙红色,另一种表现黄色；TSJ-2成熟果实着色浅于野生型。(3)质体观察显示,在野生型山金柑的衰老叶柄以及光生长条件下的珠心苗根系中,主要形成叶绿体结构,而在相应的转基因组织中主要为有色体；在野生型山金柑的花瓣,暗生长条件下的珠心苗根系和胚状体中,可以观察到明显的淀粉体类似结构,而在相应的转基因组织中可以观察到丰富的有色体。上述特征证实类胡萝卜素积累可以促进有色体发育。另外,电镜观察显示,TSJ-1黄色果实和TSJ-2果实的有色体中均含有较丰富的高电子密度质体球,但质体球数目以及有色体分布密度低于野生型果实。(4)与野生型相比,两株转基因山金柑的3年龄叶片几乎完全脱落,表现出叶片早衰现象。转基因山金柑中相对较高的ABA水平可能与叶片早衰有关。4.红夏橙DH愈伤系创制并用于LCYB1和LCYE基因的等位多态性分析本研究通过花药培养获得两份红夏橙[Citrus sinensis (L.) Osbeck]胚性愈伤系。倍性检测以及SSR标记分析表明,两份愈伤为双单倍体系(DHs)。本研究进一步利用红夏橙DHs证实类胡萝卜素代谢关键基因LCYB1和LCYE在甜橙基因组中均为杂合状态,呈现等位多态性特征。综上所述,本研究对理解柑橘以及植物类胡萝卜素代谢的生物学本质具有重要价值,并且为柑橘果实色泽改良提供了重要的基础数据,同时也为甜橙基因组测序提供了重要的材料。更多还原

【Abstract】 Carotenoids are plant indispensable secondary metabolites, involved in a series of biological processes, such as photosynthesis, antioxidation, hormone biosynthesis, and attractants for pollinators. Carotenoids are also essential health-protecting compounds for supplying provitamin A, suppressing the development of several chronic diseases, and being involved in human’s immunity, embryonic development, and reproduction. Plant is one of the major organisms containing carotenogenesis, and is also the most important carotenoid source for human life. To exert their effective usage, it is essential to understand the regulation mechanism of plant carotenoid metabolism.Citrus exhibits extensive diversity of carotenoid patterns, and is the better material for plant carotenoid metabolism study. In this study, by creating various genetic resources, including transgenic embryogenic calli, transgenic plants, and Valencia sweet orange DH lines, we studied the regulation mechanism of carotenoid metabolism, correlation between carotenoid metabolism and other biological processes, and allelic diversity of two key carotenogenic genes LCYB1and LCYE in sweet orange. The main results were as followed:1. Regulating carotenoid accumulation through overexpressing CrtB and DSM2in citrus embryogenic calli(1) We engineered embryogenic calli from Marsh grapefruit(Citrus paradise Macf.), Star Ruby grapefruit(Citrus paradise Macf.), Cara Cara navel orange [Citrus sinensis (L.) Osb.], and Sunburst mandarin [Citrus reticulata Blanco×(Citrus paradisi Macf.×Citrus Reticulate)] as Engineered Cell Models (ECMs) by overexpressing CrtB (a bacterial phytoene synthase gene). In the ECMs, the levels of total carotenoids markedly increased, and carotenoid patterns showed diversity depending on the genotypes.30free-type carotenoids were detected in the ECMs, including all of types in wild types and some new ones, such as lycopene.(2) Especially in the ECMs from dark-grown culture, there emerged a favored β,β-pathway characterized by a striking accumulation of β-carotene, which was dramatically unique from those in the wild-type calli. Based on the data in this study, a model was established to explain the favoring of β,β-pathway in the ECMs. This model suggests that LCYE (lycopene ε cyclase) plays a bottleneck role in the presence of abundant lycopene substrates leading to an altered carotenoid composition.(3) It was noted that the ECMs under irradiation showed a marked decrease in β-carotene and phytoene levels, while lutein kept stable. Most carotenoids were also reduced in37℃grown ECM line, but zeaxanthin level increased significantly. In addition, ECM exhibited unstable CrtB transcription and carotenoid accumulation, which is associated with epigenetic regulation.(4) Ripe flavedos (the colored outer layer of citrus fruits) from four consistent genotypes were offered as a comparative system to the ECMs. The comparative analysis showed ripe flavedos contained chiefly esterified violaxanthin and cryptoxanthin, or lycopene. Unlike flavedos, the ECMs did not form chromoplast, instead they sequestered most carotenoids in the amyloplasts in crystal form. Conglomerates containing abundant carotenoids were observed in the vacuoles of ECMs, suggesting an underlying catabolism mechanism that governs carotenoids.(5) Transcriptional analysis revealed that most of isoprenoid and carotenoid genes exhibited up-regualted expression in flavedos, especially HYB (gene encoding β-carotene hydroxylase) showed a markedly flavedo-specific expression, but was suppressed in the calli. Furthermore, coexpression of CrtB and DSM2(a HYD gene from rice) in the ECMs confirmed that HYD predominantly mediated the preferred carotenoid patterns between the ECMs and flavedos, and also unraveled that the carotenoid crystals in the ECMs were majorly composed of P-carotene. In addition, transcript perturbation of endogenous carotenogenic genes was observed in ECMs, especially in the transgenic callus lines of Star Ruby grapefruit.2. Engineered carotenoid accumulation affects plastid development and metabolsim(1) Transgenic callus lines with overexpressive CrtB gene (ECMs) accumulated lower levels of starch and higher levels of soluble sugars relative to wild types, showing the effect of carotenoid accumulation on carbohydrate metabolism, and also suggesting a potential chromoplast development in the ECMs. Though sucrose-free culture, we found ECMs indeed had the chromoplast development program, while it was suppressed by the predominant starch accumulation under normal culture.(2) Global transcriptional diversities between ECMs and wild types of four genotypes were investigated through citrus microarray analysis. The results showed similar diversity pattern existed in four groups, which was characterized by a marked up-regulation of abundant stress or redox response genes, such as POD (gene encoding peroxidase), GST (gene encoding glutathione S transferase), HSP (gene encoding heat shock protein) and the genes involved in phenylpropanoid metabolism. In addition, some anthocyanin genes were down-regulated in the ECMs, which suggested that carotenoid accumulation could negatively regulated anthocyanin biosynthesis. We discovered a-amylase gene up-regulated in the ECMs, which was probably related to the alteration of starch content in the ECMs.(3) ABA levels in the ECMs showed little correlation with stress and redox response. Furthermore, reactive oxidative species (ROS) were investigated, and the result showed that carotenoid accumulation could alter cellular redox status. ECMs contained lower O2-(superoxide radical) levels compared to wild types, while H2O2(hydrogen peroxide) levels were higher in the ECMs. Phenotypes of the ECM treated by concentrated sucrose and reductives suggested that O2-could be involved in the degradation of carotenoids in the ECMs. In addition, this study showed that redox status potentially affected starch metabolism in citrus callus.(4) To confirm that carotenoid accumulation could negatively regulate anthocyanin biosynthesis, an apple ECM was constructed through overexpressing CrtB gene in apple callus with anthocyanin accumulation. This ECM yielded more carotenoids than wild type, but anthocyanin biosynthesis was suppressed. Norflurazon, an inhibitor of plant carotenoid biosynthesis could partially resume anthocyanin accumulation in the apple ECM. Further transcriptional analysis showed that anthocyanin biosynthetic genes were down-regulated in this ECM.3. Transformation with CrtB gene in precocious trifoliate orange and Hongkong kumquat, and study of carotenoid accumulative characters in transgenic plants(1) Through CrtB transformation of early flowering citrus, we obtained one transgenic precocious trifoliate orange [Poncirus trifoliate (L.) Raf] line and two transgenic Hongkong kumquat (Fortunella hindsii Swingle) lines. Regenerated transgenic shoots of precocious trifoliate orange showed orange, suggesting a high carotenoid accumulation. Whereas, most of orange regenerated shoots fail to become green, and could not develop further. One regenerated transgenic precocious trifoliate orange shoot with orange to green transition was further cultured, it showed dwarf phenotype.(2) Green tissues of transgenic Hongkong kumquats had no orange phenotype, while their petals, senescent leafstalks, seeds, and roots exhibited orange, suggesting high accumulation of carotenoids. Roots of wild-type seedlings could become green under light-grown culture, while roots of transgenic seedlings kept orange. In addition, the ripe fruits of transgenic Hongkong kumquat1(TSJ-1) showed diverse color:one represented orange similar to wild type, another one represented yellow; the ripe fruits of TSJ-2exhibited a little lighter color compared with those of wild type.(3) Plastid inspection revealed that, in the cells of senescent leafstalks and light-grown roots of wild type, chloroplasts were main plastid type, while chromoplasts were observed in transgenic cells; abundant amyloplasts existed chiefly in the cells of wild-type petals, dark-grown roots and embryoids, but chromoplasts appeared predominantly in transgenic cells. All these microscopic data confirmed that carotenoid accumulation confers chromoplast development in transgenic Hongkong kumquat. In addition, the yellow fruits of TSJ-1and mature fruits of TSJ-2both contained abundant electron-dense plastoglobules, but showed lower amount of chromoplast and plastoglobules than wild-type fruits.(4) Compared to wild types, all three-year-old leaves of two transgenic Hongkong kumquats almost abscised, suggesting an early senescence symptom. Leaves of transgenic Hongkong kumquats contained higher ABA contants, which could induce the early senescence in transgenic Hongkong kumquats.4. Recovery of Rohde Red sweet orange DH line for studying allelic diversity of two carotenogenic genes LCYB1and LCYE in sweet orangeTwo callus lines of Valencia sweet orange cv. Rohde Red [Citrus sinensis (L.) Osbeck] were recovered through anther culture in this study. Ploidy investigation and SSR analysis revealed two callus lines were doubled haploids (DHs). DHs provided support for the allelic diversity of two carotenogenic genes, LCYE and LCYB, in sweet orange.In summary, this study shows a valuble approach to comprehension of the nature of carotenoid metabolism in citrus and other plants, and supplies some important basic data with regard to color improvement of citrus fruit, and also provides an essential material for citrus genome sequence.更多还原