本文關(guān)鍵詞： 玫瑰 類黃酮 花青素 原花青素 擬南芥突變體 同源基因 候選基因篩選　出處：《華中農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：玫瑰(Rosa rugosa Thunb.)是薔薇科重要觀賞植物,以其獨(dú)特的造型、絢麗多彩的花色及抗逆性強(qiáng)等特征在園林中被廣泛應(yīng)用。且玫瑰作為精油提取的重要材料,蘊(yùn)藏巨大的經(jīng)濟(jì)價(jià)值。類黃酮類化合物是植物體內(nèi)重要的次生代謝產(chǎn)物,對(duì)植物的色澤形成有著重要的影響。類黃酮類化合物具有較強(qiáng)的抗氧化性,在植物的抗逆、響應(yīng)極端外界環(huán)境變化等方面具有積極的作用。此外,有報(bào)道花青素,原花青素等類黃酮類代謝物具有消炎、活血、抗氧化等作用,還可以避免冠心病、炎癥、癌癥等疾病的發(fā)生,對(duì)人類的身體健康有著重要的意義。目前,有關(guān)類黃酮合成代謝路徑的研究比較清晰,但仍然有些謎團(tuán)尚未解開。玫瑰的研究也主要以傳統(tǒng)育種為主,分子方面的研究報(bào)道較少。隨著玫瑰轉(zhuǎn)基因體系的成熟,利用現(xiàn)代生物技術(shù)與傳統(tǒng)育種手段相結(jié)合提高玫瑰品質(zhì)成為可能。本課題組前期通過構(gòu)建薔薇科不同物種花青素代謝途徑相關(guān)的同源結(jié)構(gòu)基因共表達(dá)網(wǎng)絡(luò)和蛋白質(zhì)-蛋白質(zhì)互作網(wǎng)絡(luò)分析,初步找出28個(gè)保守基因集,進(jìn)而從擬南芥中挑選出與這28個(gè)基因集中最同源的且與花色相關(guān)的35個(gè)候選基因。之后,本課題以擬南芥35個(gè)候選基因的突變體材料為研究對(duì)象,通過系列試驗(yàn)篩選影響類黃酮代謝的相關(guān)基因,探究其功能,并進(jìn)一步研究玫瑰中同源基因功能,更深層次的探究類黃酮代謝路徑的分子調(diào)控機(jī)制,為玫瑰的育種奠定基礎(chǔ)。主要研究結(jié)果如下:1.通過純合突變體表型觀測(cè)發(fā)現(xiàn)288突變體及95突變體表型明顯,與對(duì)照組差別較大。其中288突變體蓮座葉表現(xiàn)出葉片寬、葉柄短,節(jié)間分支多等表型;而95突變體表現(xiàn)出則株型矮小,葉片圓厚、革質(zhì),花瓣數(shù)多,莢果呈短棒狀等表型。2.通過DMACA、DPBA染液對(duì)擬南芥突變體不同組織的原花青素和黃酮醇積累進(jìn)行原位快速染色分析,發(fā)現(xiàn)212、272、288、404、494、550、6506、0653等突變體黃酮醇含量較低。3.通過高效液相色譜法(HPLC)測(cè)定擬南芥突變體花序中黃酮醇含量,結(jié)果顯示,與對(duì)照組相比,272、288、404、494、550、6506擬南芥突變體花序中黃酮醇含量大量減少,呈極顯著性差異;288、403突變體花序中的黃酮醇含量明顯減少,呈顯著性差異;0653突變體花序中黃酮醇的含量則與對(duì)照組差別不大。4.0.5μM IAA和0.5μM NAA生長(zhǎng)素處理擬南芥突變體,發(fā)現(xiàn)在0.5μM IAA處理的情況下,494、550、272、403突變體根長(zhǎng)有極顯著性差異,6506有顯著性差異;在0.5μM NAA處理的情況下,550、288、403、6506突變體根長(zhǎng)呈極顯著性差異,272呈顯著性差異。5.生物信息學(xué)方法分析候選基因在擬南芥、玫瑰中同源基因的同源一致性及保守結(jié)構(gòu)域分析,發(fā)現(xiàn)候選基因高度同源,且高度保守。在擬南芥和“紫枝”玫瑰基因組中分析這些基因在不同組織部位的表達(dá)情況及表達(dá)趨勢(shì)分析,發(fā)現(xiàn)候選基因與類黃酮代謝路徑中部分關(guān)鍵酶基因表達(dá)趨勢(shì)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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S685.12

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