蔷薇科植物类黄酮合成相关候选基因的筛选

发布时间：2018-03-02 07:21

本文关键词： 玫瑰类黄酮花青素原花青素拟南芥突变体同源基因候选基因筛选　出处：《华中农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：玫瑰(Rosa rugosa Thunb.)是蔷薇科重要观赏植物,以其独特的造型、绚丽多彩的花色及抗逆性强等特征在园林中被广泛应用。且玫瑰作为精油提取的重要材料,蕴藏巨大的经济价值。类黄酮类化合物是植物体内重要的次生代谢产物,对植物的色泽形成有着重要的影响。类黄酮类化合物具有较强的抗氧化性,在植物的抗逆、响应极端外界环境变化等方面具有积极的作用。此外,有报道花青素,原花青素等类黄酮类代谢物具有消炎、活血、抗氧化等作用,还可以避免冠心病、炎症、癌症等疾病的发生,对人类的身体健康有着重要的意义。目前,有关类黄酮合成代谢路径的研究比较清晰,但仍然有些谜团尚未解开。玫瑰的研究也主要以传统育种为主,分子方面的研究报道较少。随着玫瑰转基因体系的成熟,利用现代生物技术与传统育种手段相结合提高玫瑰品质成为可能。本课题组前期通过构建蔷薇科不同物种花青素代谢途径相关的同源结构基因共表达网络和蛋白质-蛋白质互作网络分析,初步找出28个保守基因集,进而从拟南芥中挑选出与这28个基因集中最同源的且与花色相关的35个候选基因。之后,本课题以拟南芥35个候选基因的突变体材料为研究对象,通过系列试验筛选影响类黄酮代谢的相关基因,探究其功能,并进一步研究玫瑰中同源基因功能,更深层次的探究类黄酮代谢路径的分子调控机制,为玫瑰的育种奠定基础。主要研究结果如下:1.通过纯合突变体表型观测发现288突变体及95突变体表型明显,与对照组差别较大。其中288突变体莲座叶表现出叶片宽、叶柄短,节间分支多等表型;而95突变体表现出则株型矮小,叶片圆厚、革质,花瓣数多,荚果呈短棒状等表型。2.通过DMACA、DPBA染液对拟南芥突变体不同组织的原花青素和黄酮醇积累进行原位快速染色分析,发现212、272、288、404、494、550、6506、0653等突变体黄酮醇含量较低。3.通过高效液相色谱法(HPLC)测定拟南芥突变体花序中黄酮醇含量,结果显示,与对照组相比,272、288、404、494、550、6506拟南芥突变体花序中黄酮醇含量大量减少,呈极显著性差异;288、403突变体花序中的黄酮醇含量明显减少,呈显著性差异;0653突变体花序中黄酮醇的含量则与对照组差别不大。4.0.5μM IAA和0.5μM NAA生长素处理拟南芥突变体,发现在0.5μM IAA处理的情况下,494、550、272、403突变体根长有极显著性差异,6506有显著性差异;在0.5μM NAA处理的情况下,550、288、403、6506突变体根长呈极显著性差异,272呈显著性差异。5.生物信息学方法分析候选基因在拟南芥、玫瑰中同源基因的同源一致性及保守结构域分析,发现候选基因高度同源,且高度保守。在拟南芥和“紫枝”玫瑰基因组中分析这些基因在不同组织部位的表达情况及表达趋势分析,发现候选基因与类黄酮代谢路径中部分关键酶基因表达趋势一致。
[Abstract]:Rosa rugosa Thunb.is an important ornamental plant of Rosaceae, which is widely used in gardens with its unique shape, colorful flowers and strong resistance to stress. Flavonoids are important secondary metabolites in plants and have an important effect on the formation of plant color. Flavonoids have strong antioxidant properties and are resistant to stress in plants. In addition, it has been reported that flavonoid metabolites such as anthocyanins and procyanidins have anti-inflammatory, blood-activating and antioxidant effects, and can also prevent coronary heart disease and inflammation. The occurrence of diseases such as cancer is of great significance to the health of human beings. At present, the research on the pathway of flavonoid biosynthesis is clear, but there are still some mysteries to be solved. The research on roses is mainly based on traditional breeding. There are few reports of molecular studies. As the rose transgenic system matures, It is possible to improve the quality of rose by combining modern biotechnology with traditional breeding methods. The coexpression network and protein of homologous structure genes related to anthocyanin metabolic pathway of different species of Rosaceae were constructed in the early stage of our study. Qualitative protein interaction network analysis, A total of 28 conserved gene sets were preliminarily identified and 35 candidate genes most homologous to the 28 loci were selected from Arabidopsis thaliana. Subsequently, the mutants of 35 candidate genes in Arabidopsis thaliana were studied. Through a series of experiments to screen the related genes affecting flavonoid metabolism, explore its function, and further study the function of the homologous genes in rose, and further explore the molecular regulation mechanism of flavonoid metabolic pathway. The main results of this study are as follows: 1.Through the observation of homozygous mutants, we found that 288 mutants and 95 mutants were obviously different from the control group. The rosette leaves of 288 mutants showed wide leaves and short petioles. While 95 mutants showed short plant type, thick round leaves, leathery leaves, and many petals. The accumulation of procyanidins and flavonols in different tissues of Arabidopsis thaliana mutant was analyzed by in situ rapid staining. It was found that the flavonol content in the inflorescence of Arabidopsis thaliana mutant was determined by high performance liquid chromatography (HPLC). The results showed that the flavonol content in the inflorescence of Arabidopsis thaliana mutant was much lower than that of the control group. There was significant difference in flavonol content in the inflorescences of mutants 288tai403, while the contents of flavonols in the inflorescence of mutants 6053 were not different from those of the control group. 4.0.5 渭 M IAA and 0.5 渭 M NAA auxin were used to treat Arabidopsis mutants. It was found that there was a significant difference in root length between two mutants treated with 0. 5 渭 M IAA. Under the condition of 0.5 渭 M NAA treatment, there was a significant difference in root length between two mutants. 5. bioinformatics method was used to analyze the homology and conserved domain of candidate genes in Arabidopsis thaliana and rose. The candidate genes were found to be highly homologous and highly conserved. The expression and expression trend of these genes in different tissues were analyzed in Arabidopsis thaliana and Violet Rosa Rosa genome. It was found that the expression trend of candidate genes was consistent with that of some key enzyme genes in flavonoid metabolic pathway.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S685.12

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