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| 红砂类黄酮生物合成途径关键酶黄烷酮 3-羟化酶(F3H)基因响应环境胁迫的调控 | |
| 其他题名 | Regulation of flavonone 3-hydroxylase (F3H) involved in flavonoids biosynthesis pathway in response to environmental stresses in Reaumuria soongorica |
| 刘美玲 | |
| 出版年 | 2012 |
| 学位类型 | 硕士 |
| 导师 | 刘玉冰 |
| 学位授予单位 | 中国科学院大学 |
| 中文摘要 | 类黄酮是植物中普遍存在的一类天然多酚类化合物，是植物为适应复杂多变的自然环境，在长期进化过程中形成的一类重要的次生代谢产物。在植物吸引昆虫传粉、花粉管伸长、抵抗紫外辐射及其他逆境胁迫中发挥着重要的作用。红砂(Reaumuria soongorica)是一种在中国的西北荒漠地区分布较广，且抗逆性极强的荒漠植物，并且红砂叶片和茎提取物中含有丰富的类黄酮。本文以荒漠植物红砂为材料，克隆了类黄酮生物合成途径的关键酶黄烷酮3-羟化酶(flavonone 3-hydroxylase, F3H)基因的全长cDNA序列，并对增强UV-B辐射和干旱两种胁迫下F3H基因参与的类黄酮生物合成途径的调控进行了分析。通过实时定量 RT-PCR方法研究了不同胁迫下F3H基因在转录水平的变化；分析了类黄酮生物合成途径中关键酶黄烷酮-3羟化酶(flavonone 3-hydroxylase, F3H)和二氢黄酮醇4-还原酶(dihydroflavonol 4-reductase, DFR)的活性及代谢产物总黄酮与花青素的含量变化，以及红砂类黄酮的抗氧化活性；将类黄酮生物合成途径的响应与不同胁迫下的膜脂过氧化程度(以MDA含量表示)和光合色素的变化进行相关性分析，揭示植物次生代谢产物类黄酮生物合成途径响应环境胁迫的调控机理。研究结果如下：\n(1)获得F3H基因的全长cDNA序列，共1409bp，其中包含1101bp的开放阅读框(90bp-1190bp之间)，命名为RsF3H，在GenBank中的登录号为JQ043380。该阅读框编码一个由366个氨基酸组成的蛋白质，分子量为41.35184KD,等电点为5.011。该序列的系统进化分析表明：RsF3H与陆地棉(GhF3H)同源性最高，为84.2%，其次与海岛棉(GbF3H)、龙眼(DlF3H)、苦荞麦(FtF3H)、茶树(CsF3H)的同源性也很高，分别分别为：83.9%、83.7%、83.2%和82.4%。编码蛋白的二级结构包含了35.8% a螺旋、24% b折叠和29.5%任意卷曲，保守结构域查找显示RsF3H蛋白质含有一个高度保守区域20G-FeⅡ-Oxy。\n(2)不同胁迫下实时定量RT-PCR分析RsF3H基因转录水平的变化结果表明：增强UV-B辐射和干旱胁迫处理过程中RsF3H基因转录水平均表现出先升高后降低的趋势，但最终仍高于处理前的水平。说明增强UV-B辐射和干旱胁迫均可以诱导F3H基因的表达。\n(3)两种环境胁迫下类黄酮生物合成途径关键酶F3H、DFR酶活性和代谢产物总黄酮和花青素含量的测定结果显示，处理期间F3H活性均先升高后降低，但仍高于处理前水平；DFR活性与花青素含量随处理时间延长逐渐升高。增强UV-B辐射下，总黄酮含量先降低后升高；干旱胁迫下总黄酮含量随处理时间延长逐渐增加。说明环境胁迫对类黄酮生物合成途径有诱导作用。\n(4)总黄酮对有机自由基DPPH的清除率及抑制不饱和脂肪酸氧化为MDA的能力表明：两种胁迫下总黄酮对DPPH清除率及对MDA形成的抑制率均随处理时间的延长增加，总黄酮的抗氧化能力增强。\n \n(5)不同胁迫处理期间MDA含量都随着胁迫时间的延长迅速增加，表明植物细胞的膜脂过氧化水平增加。光合色素叶绿素含量随处理时间的延长而减少，但Chla/Chlb、Car/Chl增加。类黄酮生物合成途径的响应与MDA和光合色素变化的相关性分析表明：DFR活性、花青素含量、总黄酮对DPPH清除率和对MDA形成的抑制率均与MDA以及光合色素存在显著或极显著的相关性，表明次生物质类黄酮生物合成途径参与了植物的抗逆过程，通过发挥黄酮类物质的抗氧化功能，提高红砂在逆境胁迫下的自我保护能力。 |
| 英文摘要 | Flavonoids are ubiquitous plant secondary metabolites which function in the attraction of pollinators, elongation of pollen tube and protectants against Ultraviolet light and other adverse environmental conditions. Reaumuria soongoricais a desert plant species that is mainly distributed in desert areas of northwestern China, and it can survive under severe environmental stresses. There are abundant flavonoids in the leaves and items of R. soongorica. To investigate the molecular mechanisms of stress tolerance in R. soongorica, we cloned the full length cDNA of flavonone 3-hydroxylase (F3H) gene from R. soongorica, and then studied the regualtion of flavonoids biosynthesis pathway in response to enhanced UV-B radiation and drought stresses. The transcriptional levels of F3H gene were investigated by real-time quantitative PCR. The activity of key enzymes F3H and DFR (dihydroflavonol 4-reductase) involved in flavonoids pathway were assayed by RS-HPLC-MS. The contents of total flavonoids and anthocyanins and the antioxidant ability of total flavonoids under stresses were also determined. And we analyzed the correlations between flavonoids biosynthesis pathway and the content of MDA and photosynthesis pigments. The main results are as follows:\n(1) We successfully isolated a full length cDNA of F3H gene from R. soongorica which was 1409bp with an open reading frame (ORF) of 1101bp and was named as RsF3H (Genbank accession No. JQ043380). RsF3H is predicted to encode a 41.35KD protein which consists of 366 amino acid residues, and the PI of the predicted prptein is 5.011. The phylogenetic tree suggested that the RsF3H is most homologous to the Gossypium hirsutum GhF3H (84.2%), Gossypium barbadense GbF3H(83.9%), Dimocarpus longan DlF3H(83.7%), Fagopyrum tataricum FtF3H(83.2%), Camellia sinensis CsF3H(82.4%). The secondary structure of the putative RsF3H protein was analyzed by SOPMA. The result showed that the putative RsF3H peptide contained 35.8% alpha helix，24% beta turn and 29.5% random coil. The conserved domain search result showed that the putative protein contained a high conserved domain: 20G-FeⅡ-Oxy.\n(2) The transcriptional levels of RsF3H gene under stresses were analyzed by real-time quantitative PCR. The results showed the relative expression of F3H gene were higher than that of untreated. It is suggested that the F3H gene can be induced by enhanced UV-B radiation and drought stresses.\n(3) The regulation of flavonoids biosynthesis pathway under stresses was investigated. The activity of F3H increased within 6d and 4d under enhanced UV-B radiation and drought stress, respectively, and then dropped to a level which was still higher than that of untreated. The activity of DFR and anthocyanins content increased during treatment periods. The total flavonoids content reduced within 4d, and then increased under UV-B radiation. It, however, showed a steady increasing trend under drought stress. It can be concluded that both stresses could induce the up-regulation of flavonoids biosynthesis pathway.\n(4) We assessed the antioxidant ability of total flavonoids by determining the rate of scavenging DPPH and inhibition of MDA of total flavonoids. The results showed the antioxidant ability increased during the treatment periods.\n(5) The increase of MDA content suggested that the membranes of plant were damaged under stresses. The contents of Chla, Chlb and Chl decreased as treatment time increased, but the ratios of Chla/Chlb and Car/Chl increased. The correlation between flavonoids biosynthesis pathway and the contents of MDA and photosynthesis pigments were analyzed. The DFR activity, anthocyanins content and antioxidant ability of flavonoids has significant correlation with MDA content and photosynthesis pigments. It is suggested that flavonoids play an antioxidant role in protecting plant from damaging by stresses. |
| 中文关键词 | 红砂 ; 类黄酮 ; 黄烷酮3-羟化酶 ; 环境胁迫 ; 生物合成途径 |
| 英文关键词 | Reaumuria soongorica flavonoids flavonone 3-hydroxylase gene cloning UV-B radiation drought stress biosynthesis pathway |
| 语种 | 中文 |
| 国家 | 中国 |
| 来源学科分类 | 生态学 |
| 来源机构 | 中国科学院西北生态环境资源研究院 |
| 资源类型 | 学位论文 |
| 条目标识符 | http://119.78.100.177/qdio/handle/2XILL650/287086 |
| 推荐引用方式 GB/T 7714 | 刘美玲. 红砂类黄酮生物合成途径关键酶黄烷酮 3-羟化酶(F3H)基因响应环境胁迫的调控[D]. 中国科学院大学,2012. |
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