【作者】 张超；

【导师】 董丽；

【作者基本信息】 北京林业大学 ， 园林植物与观赏园艺， 2014， 博士

【摘要】 花色是观赏植物最重要的观赏性状之一。花青素苷是被子植物花色表达最主要的色素物质。对模式植物的研究表明糖不仅为花青素苷合成提供代谢底物,还以信号分子形式调控花青素苷合成调节基因和结构基因的表达。牡丹(Paeonia suffruticosa)是我国传统名花之一,从古至今一直深受我国人民的喜爱。了解外源糖对牡丹切花花色和花青素苷合成的调控机制,可为牡丹切花采后保鲜技术的开发提供理论依据。本研究选取紫红色品种‘洛阳红’切花为试材,对葡萄糖调控牡丹切花‘洛阳红’花青素苷合成的分子机理进行了研究。同时,本研究还分离了牡丹葡萄糖信号感知和信号转导关键因子己糖激酶(Hexokinase, HXK)基因家族成员,并对其生物学功能进行揭示。相关结果如下：(1)牡丹‘洛阳红’切花花色品质不如在体花朵,切花花瓣中花青素苷含量和可溶性糖含量低于在体花朵。外源60、90、120g·L-1葡萄糖处理增加切花花瓣中可溶性糖含量和花青素苷含量,并改善‘洛阳红’切花的花色品质,表现为降低花色明度、增加花色红度和彩度。(2)构建了牡丹‘洛阳红’切花混合花瓣转录组,利用Illumina平台测序得到57,206,122条原始reads,经数据过滤和组装生成50,829条unigeneso根据unigene信息分析,ANS基因有1条unigene, CHS、CHI、F3H、F3’H、DFR、MYB、bHLH和WD40等花青素苷合成相关基因有多条unigenes,推测这些基因在牡丹‘洛阳红’中可能以多基因家族形式存在,同时还推测牡丹‘洛阳红’中可能存在6个HXK基因家族成员。(3)基于牡丹‘洛阳红’花瓣转录组unigenes序列,分离得到牡丹花青素苷合成5个调节基因和6个结构基因序列。系统进化树分析,PsbHLHl、PsbHLH3、 PsWD40-1、PsWD40-2和PsMYB2基因编码的蛋白可能参与调控牡丹类黄酮和花青素苷的生物合成。PsCHIl基因在叶片中表达量最高,而其他10个花青素苷合成相关基因在花器官相关组织(花瓣、雄蕊、雌蕊和萼片)中高丰度表达。PsbHLH3、PsWD40-1、 PsWD40-2、PsMYB2、PsCHS1、PsF3Hl和PsDFRl基因的低丰度表达可能是导致牡丹切花‘洛阳红’花瓣中花青素苷含量降低,花瓣颜色变浅的原因。(4)3-O-甲基葡萄糖(不能被HXK磷酸化的葡萄糖类似物)处理短时间内促进牡丹‘洛阳红’切花花青素苷含量和PsbHLH1、PsbHLH3、PsWD40-1、PsWD40-2、 PsMYB2、PsCHSl和PsCHI1基因的表达量,表明短时间渗透压的改善促进牡丹‘洛阳红’切花花青素苷合成。调节基因PsWD40-2、PsMYB2和结构基因PsCHSl、PsCHI1、 PsF3’H1响应葡萄糖信号调控。其中,葡萄糖信号通过不依赖HXK途径调控PsWD40-2、PsMYB2、PsCHS1和PsCHIl基因的表达,同时通过依赖HXK途径调控PSF3’H1基因的表达。PsF3’H1基因表达量与切花花青素苷含量紧密相关,被认为是葡萄糖调控牡丹‘洛阳红’切花花青素苷合成的关键基因。甘露糖(能被HXK磷酸化的葡萄糖类似物)处理持续上调多个基因的表达,下调PsF3’H1和PsANS1基因的表达,严重抑制花瓣中花青素苷的积累。(5)与葡萄糖处理‘洛阳红’切花相比,相同摩尔浓度蔗糖处理切花花青素苷含量较低,蔗糖处理切花PsWD40-1、PsWD40-2、PsCHS1、PSF3H1、PsDFR1、PSANS1基因表达量显著高于葡萄糖处理,而PsCHI1和PsF3’H1基因表达量显著低于葡萄糖处理。(6)从牡丹中分离得到葡萄糖信号感知和转导关键因子HXK的2个同源基因PsHXK1和PsHXK2的cDNA全长。依据序列分析和系统进化树分析,推测PsHXK1和PsHXK2蛋白均具有催化己糖磷酸化的功能。PsHXK1基因在花瓣中表达量最高,而PsHXK2基因在雄蕊中表达量最高。根据牡丹‘洛阳红’花瓣中PsHXK1和PsHXK2基因表达情况,推测这2个基因的转录水平可能与己糖含量有密切的关联。PSHXK1和PsHXK2基因过表达转化拟南芥的研究发现,PsHXK1和PsHXK2具备糖信号感知和转导的功能,并响应高浓度葡萄糖调控花青素苷合成相关基因的表达,提高转基因拟南芥的花青素苷含量。上述研究结果表明,除了渗透作用和代谢作用,葡萄糖促进牡丹切花‘洛阳红’花青素苷积累,主要依靠其信号转导调控。此外,PsHXK1和PsHXK2在葡萄糖调控花青素苷合成过程中发挥着重要的作用。上述结论初步解析了葡萄糖调控牡丹切花花青素苷合成的分子机理,为牡丹切花采后保鲜技术的开发提供理论依据。更多还原

【Abstract】 Flower color is one of the important traits of ornamental plants. Anthocyanin is the main pigment for flower coloration of angiosperms. According to the studies of model plants, sugars not only provide metabolism substrate for anthocyanin biosynthesis, but also regulate the expression of anthocyanin biosynthetic regulatory genes and structural genes through sugar signalling. Tree peony(Paeonia suffruticosa), a Chinese traditional ornamental plant, is greatly appreciated by Chinese from ancient to now. Understanding the regulation of exogenous sugar on flower color and anthocyanin biosynthesis of tree peony cut flowers would provide theoretical foundation for the postharvest technology development of tree peony cut flowers. Cut flowers of a purple red cultivar’Luoyang Hong’of tree peony were used as the materials in this study to reveal the molecular mechanism of the involvement of glucose in improving the flower color and regulating the anthocyanin biosynthesis. What is more, gene family members of hexokinase (HXK), a key protein involved in sugar sense and signaling, were isolated in tree peony and their biological functions were further studied. The results are showed as follows:(1) The quality of flower color of cut flower is inferior to that of on-tree flower of tree peony ’Luoyang Hong’. Anthocyanins content and soluble sugar content of cut flowers are lower than those of on-tree flowers. Exogenous60,90and120g-L"1glucose enhance soluble sugar content and anthocyanins content in petals, and also improve petal color quality of cut tree peony’Luoyang Hong’ with lower lightness and higher redness and chroma.(2) The transcriptome from mixed petals of cut tree peony’Luoyang Hong’ was contructed and sequenced using the Illumina platform. A total of57,206,122raw reads were obtained. After data filtering and sequence assembly,50,829unigenes were generated. Based on the analysis of unigene information, ANS gene has one unigene, while other anthocyanin biosynthetic genes CHS, CHI, F3H, F3’H, DFR, MYB, bHLH and WD40exist in the genome of tree peony ’Luoyang Hong’ as multigene family. It is predicted that tree peony’Luoyang Hong’ probably has6HXK gene family members.(3) Based on the unigene sequences of petal transcriptome of tree peony’Luoyang Hong’,5regulatory genes and6structural genes of anthocyanin biosynthesis from tree peony were isolated. Phylogenetic tree analysis reveals that the proteins encoded by PsbHLHl, PsbHLH3, PsWD40-1, PsWD40-2and PsMYB2are probably involved in the regulation of flavonoid and anthocyanin biosynthesis in tree peony. PsCHIl is highly expressed in leaves, but other10anthocyanin biosynthetic genes are highly expressed in the flower-related tissues (petals, stamens, carpels and sepals). Low expression levels of PsbHLH3, PsWD40-1, PsWD40-2, PsMYB2, PsCHSl, PsF3H1and PsDFRl may be responsible for less anthocyanins content and color fading in cut tree peony’Luoyang Hong’.(4) Treatment of3-O-methylglucose (a kind of glucose analog which can’t be phosphorylated by hexokinase) enhances anthocyanins content and induces the expression levels of PsbHLHl, PsbHLH3, PsWD40-1, PsWD40-2, PsMYB2, PsCHS1and PsCHI1in short time, suggesting that short time improvement of osmotic pressure induces the anthocyanin biosynthesis of cut tree peony’Luoyang Hong’. Glucose signal induces the expression levels of regulatory genes PsWD40-2, PsMYB2and structural genes PsCHSl, PsCHI1, PsF3’H1. Among them, PsWD40-2, PsMYB2, PsCHSl and PsCHIl are regulated by glucose signal via HXK-independent pathway, while PsF3’H1is regulated by HXK-dependent glucose signaling pathway. Expression level of PsF3’H1shows a close relationship to the anthocyanins content of cut tree peony ’Luoyang Hong’, so PsF3’H1is regarded as the key gene involved in the regulation of glucose on the anthocyanin biosynthesis of cut tree peony’Luoyang Hong’. Treatment of mannose, known to be phosphorylated by HXK, constantly up-regulated the expression of several anthocyanin biosynthetic genes, down-regulated the expression of PsF3’H1and PsANSl, and greatly inhibited anthocyanin accumulation in petals.(5) Anthocyanins content of cut tree peony ’Luoyang Hong’ with the treatment of the same molar concentration sucrose is lower than that of glucose-treated cut flowers. The expression levels of PsWD40-1, PsWD40-2, PsCHSl, PsF3H1, PsDFR1and PsANS1in sucrose-treated cut tree peony flowers are significantly higher than those in glucose-treated cut tree peony flowers, while the expression levels of PsCHIl and PsF3’H1in sucrose-treated cut tree peony flowers are lower than those in glucose-treated cut tree peony flowers.(6) Full-lenth cDNA sequences of PsHXK1and PsHXK2, the homologous genes of HXK gene involved in hexose sense and siganlling, were isolated from tree peony in this present study. Based on sequence analysis and phylogenetic tree analysis, it is predicted that PsHXK1and PsHXK2have the catalytic function of hexose phosphorylation. PsHXK1is highly expressed in the petals, while PsHXK1is highly expressed in the stamens. It is also predicted that the expression levels of PsHXK1and PsHXK2are probably associated with the hexoses content in the petals of tree peony’Luoyang Hong’. Overexpression of PsHXK1and PsHXK2in Arabidopsis thaliana reveals that PsHXK1and PsHXK2have the ability of sensing and transducting sugar signal and overexpression of PsHXK1and PsHXK2enhance anthocyanins content of Arabidopsis under high concentration of glucose condition through the up-regulation of the expression of several anthocyanin biosynthetic genes.The above results indicated that, besides osmotic effect and metabolic effect, glucose promotion of anthocyanin accumulation is mainly attributed to the regulation of glucose signalling. Furthermore, PsHXK1and PsHXK2play essential roles in the regulation of glucose on anthocyanin biosynthesis. Above conclusions should contribute to a better understanding of molecular mechanism of glucose regulating anthocyanin biosynthesis in cut tree peony, and would provide theoretical foundation for the postharvest technology development of cut tree peony.更多还原