【作者】 周晋；

【导师】 李顺祥； 卢向阳；

【作者基本信息】 湖南农业大学 ， 生物化学与分子生物学， 2013， 博士

【摘要】 芪类(Stilbenes)是具有均苯二烯母核或其聚合物的一类物质的总称。白藜芦醇、氧化白藜芦醇和桑皮苷是具有代表性的芪类成分,均存在于桑(Morus albaL.)等植物中。由于芪类在植物生理学、药物学和在人类健康中具有重要的研究和开发价值而被广泛研究和关注。但是芪类物质为植物次生代谢物,含量低,且大多不稳定,容易在提取、纯化过程中损失,给大规模的生产和应用带来了一定的影响,目前芪类化合物的价格还比较高。我国的桑资源丰富,但是对于桑中芪类的研究报道较少。本文以桑为研究对象,对不同品种、同一品种不同取材部位、不同采收季节的桑中主要芪类化合物的累积变化规律进行了研究。以桑幼苗为材料,研究了紫外照射后相关酶芪合酶STS、查尔酮合成酶CHS基因的表达水平和芪类、黄酮类的累积变化规律。主要研究结果如下：1.建立了HPLC-UV同时检测桑中3个芪类化合物的方法,通过梯度洗脱于21min内可完全分离,且线性关系、精密度与重复性良好,桑样品提取液在12h内稳定。方法准确、可靠、稳定,样品预处理简单,可作为桑样品中白藜芦醇、氧化白藜芦醇和桑皮苷等芪类成分的含量检测方法。2.不同品种、同品种不同取材部位、不同采收季节的桑中主要芪类物质含量存在差异。芪类物质含量相对较高的优势桑品种有红皮湖桑、中生火桑、黑鲁桑、塘10,小鸡冠桑等；含量高的部位为根的各个部位、皮部以及周皮痕迹；含量高的季节为秋冬与春季。芪类成分检测结果可作为筛选芪类物质含量高的桑树资源的依据。3.对桑树中各部位之间及与主根根皮芪类成分含量进行相关与逐步回归分析,结果显示,3个芪类成分含量大部分显著相关。按照逐步回归的方法,分别得到桑皮苷A、氧化白藜芦醇、白藜芦醇桑树各部位与主根根皮含量的回归方程,呈显著的正相关,这说明可以根据桑树其他部位芪类成分含量估测主根根皮中芪类成分含量。4.以桑幼苗为材料进行研究,UV胁迫增加了桑幼苗组织中的过氧化氢含量,使机体的活性氧不断增加,激发机体的胁迫应答机制：芪类含量增加,总抗氧化能力升高,GSH还原过氧化物,清除体内的白由氧。使植物体内活性氧的产生和清除之间趋于平衡状态,维持机体组织原有的平衡。5.以桑幼苗为研究对象,比较桑苗中对照组和紫外照射组芪类黄酮类成分的累积变化规律,以及分析紫外照射对其相关生物合成关键酶基因STS和CHS表达的影响。紫外可以诱导STS、CHS基因的表达与芪类、黄酮类化合物含量的累积,且芪类、黄酮类化合物含量与STS、CHS基因表达变化规律基本一致。说明紫外诱导后芪类、黄酮类化合物的合成与累积受到芪合酶、查尔酮合成酶基因的调控。6.测试了桑苗发育过程中受到UV诱导时不同部位STS、CHS基因的农达水平。结果表明,桑苗受到UV诱导时STS、CHS基因表达受到影响,根、茎、叶中的表达均增加,且在叶中表达变化最大。以上可知,不同品种、同一品种不同取材部位、不同采收季节的桑中主要芪类物质含量存在差异；应用相关与逐步回归分析,可以根据桑树其他部位芪类成分含量估测主根根皮中芪类成分含量。可为筛选富含芪类物质桑树资源及优势桑品种选育提供一定的实验依据。紫外诱导促进桑苗中芪合酶STS、查尔酮合成酶CHS基因的表达及芪类、黄酮类成分的累积；紫外诱导后桑苗根、茎、叶部位的STS、CHS基因表达均增加,且在叶中表达变化最大。从分子水平上初步研究了紫外诱导对桑苗中STS。 CHS基因表达和芪类、黄酮类合成的调控,为将来利用基因工程方法调控STS、 CHS基因表达与芪类、黄酮类的合成提供了理论依据和实验基础。更多还原

【Abstract】 Stilbenes are a group of materials that have the benzene diene nucleus or the polymers, including resveratrol, oxyresveratrol and mulberroside A, which can be obtained from mulberry. Stilbenes have been widely studied in plant physiology, pharmacology and human health for their important value in the research and development. But stilbenes are secondary metabolites in plant; with low content and unstable nature, they are easily lost in the process of extraction and purification. This resulted in the difficulty to produce them in a large scale and hence impaired their application, and also led to a relatively high price. Tough Mulberry resources are rich in our country; the study on stilbenes in mulberry was rare. This study aimed to investigate the composition of major stilbenes in different portions of different mulberry varieties collected in different seasons. The expression patterns of STS, CHS and the content of stilbenes, flavonoids in mulberry seedlings treated with UV light were studied. The results were as follows:1. A simple, sensitive and specific HPLC method was developed for simultaneous determination of the three major active constituents in mulberries, namely mulberroside A, oxyresveratrol, resveratrol, which were completely separated within21min, and were stable in12h, suggesting that the method can be used to analyze the three major stilbenes in mulberry.2. The main contents of stilbenes varied according to different portions of different mulberry varieties collected in different seasons. It was found that mulberry varieties, Hongpihusang, Zhongshenghuosang, Heilusang, Tang10, Xiaojiguansang have high content of stilbenes; Among different positions of mulberry, the root, bark and periderm vestige have higher level of stilbenes; the same is true for autumn, winter and spring.3. By correlation and regression analysis of the contents of stilbenes in different parts and axial root bark, we found that the contents of mulberroside A, oxyresveratrol, resveratrol in different parts and axial root bark were positively correlated. 4. The content of H2O2in mulberry seedlings treated with UV light was significantly higher than that in the control. The body defense systems response to stress by increasing the stilbenes and the total antioxidant capacity. The GSH reduces H2O2and clear away free oxygen from the body, maintaining in plants a balanced and dynamic state between the production and elimination of activated oxygen.5. The expression pattern of STS, CHS and the content of stilbenes, flavonoids in mulberry seedlings treated with UV light were studied. It was observed that the UV light induces STS, CHS expression and the accumulation of stilbenes and flavonoids in mulberry seedlings. The expression pattern of STS, CHS and that of the accumulation of stilbenes, flavonoids were basically similar, implying that stilbenes and flavonoids biosynthesis and accumulation may be regulated by STS and CHS.6. In the current study we also observed that the UV light treatment induces STS and CHS expression in root, stem and leaf of mulberry seedlings. This effect is more prominent in leaf.In conclusion, the main contents of stilbenes varied in different portions of different mulberry varieties collected in different seasons; the results of the correlation and regression analysis indicated that the contents of stilbenes in different parts could predict the content of the axial root bark. The results can be used as a reference to screen mulberry materials with higher level of stilbenes and for breeding and preserving excellent germplasm during the process of system breeding for mulberry rich in stilbenes.It was observed that the UV light treatment promoted STS, CHS expression and the accumulation of stilbenes and flavonoids in mulberry seedlings. It was also observed that UV light treatment promoted STS and CHS expression in root, stem and especially in leaf of mulberry. Our preliminary results demonstrated at the molecular level the regulation mechanism of the STS, CHS expression and the biosynthesis of stilbenes, flavonoids in mulberry seedlings treated with UV light. The results obtained during this research project provide a theoretical and experimental basis for the future study in the regulation of the expression of STS, CHS and the controlling of the biosynthesis of stilbenes, flavonoid by genetic engineering method.更多还原