玫瑰类黄酮合成相关基因的克隆和功能解析

发布时间：2018-04-26 14:26

本文选题：玫瑰 + DFR　；参考：《华中农业大学》2016年博士论文

【摘要】：玫瑰(Rosa rugosa Thunb.)是一种具有重要经济价值的观赏植物,其花色艳丽、香气溢人。玫瑰花瓣是提取香料的重要材料,玫瑰精油因价格高产量低,被人们誉为液体黄金。除此之外,玫瑰花还具有一定的消炎,活血,抗氧化等。然而目前对玫瑰的研究主要局限栽培生理和新品种选育上,随着玫瑰转基因体系的成熟,利用生物技术提高玫瑰产品附加值成为可能。本课题以玫瑰为材料研究类黄酮合成的分子机制。我们从玫瑰花瓣中分离得到二氢黄酮醇还原酶(Rr DFR1)、黄酮醇合成酶(Rr FLS1)和花青素还原酶(Rr ANR)并对它们的功能进行了深入研究。主要研究结果如下:1通过对玫瑰、野蔷薇、桃、香石竹、杜鹃、山茶以及矮牵牛7个物种的红花白花进行色素成分分析,发现红花和白花中都能检测到黄酮醇,而花青素在白花中几乎检测不到。通过对红花白花中类黄酮合成相关基因的表达分析,表明FLS和DFR的竞争性表达与花青素和黄酮醇的积累有关。为了进一步阐明这种调控规律的作用,我们从玫瑰、矮牵牛和桃中分离了FLS基因,从玫瑰和矮牵牛中分离DFR基因。烟草转基因结果表明,超表FLS能够增强黄酮醇的合成,减少花青素的积累,从而使转基因烟草花色由红色变为白色;相反,超量表达DFR基因,抑制内源Nt FLS的表达,促进花青素合成。基于上述结果,我们认为FLS和DFR对共同底物的竞争,分别实现黄酮醇和花青素的合成,从而决定红白花颜色的分化。2利用FPNI-PCR技术从玫瑰中分离得到Rr ANR,构建超量表达载体转化烟草。转基因烟草花色变白,原花青素和ABA含量增加,抗氧化能力增强。通过对Rr ANR转基因烟草和未转基因烟草转录组测序结果的分析,结果表明转基因烟草中有1586个基因上调表达,其中包括相关抗逆基因和次生代谢物合成的相关基因,特别是ABA途径和抗氧化酶系统。为了进一阐明Rr ANR提高植物抗逆性的分子机理,从烟草中分离了Nt ABF。超表达Nt ABF显著提高植株抗逆性以及抗氧化酶相关基因的表达。外源PA和ABA处理同样可以上调Nt ABF和抗氧化酶相关基因的表达。上述结果表明超量表达Rr ANR提高植物抗性是通过活性氧的清除和ABA信号实现的。
[Abstract]:Rosa rugosa Thunb. is an important ornamental plant with an important economic value. The rose petals are an important material for the extraction of spices. Rose essential oil is regarded as a liquid gold because of high price and low yield. In addition, the rose also has some anti-inflammatory, blood activating, antioxidant and so on. However, rose is currently on the rose. The research mainly limited the cultivation physiology and the selection of new varieties. With the maturity of the rose transgenic system, it is possible to use biotechnology to increase the added value of the Rose products. This topic studies the molecular mechanism of the flavonoid synthesis by the rose as the material. We isolated the two flavonol reductase (Rr DFR1) from the rose petals, and the synthesis of flavonol Enzyme (Rr FLS1) and anthocyanin reductase (Rr ANR) and their functions are deeply studied. The main results are as follows: 1 through the analysis of the pigment composition of rose, Rosa, peach, carnation, Rhododendron, camellia and Petunia, the flavonols can be detected in red flower and white flower, and anthocyanin is in white flower. The expression of the flavonoid synthesis related genes in safflower white flowers showed that the competitive expression of FLS and DFR was related to the accumulation of anthocyanin and flavonol. In order to further clarify the role of this regulation, we separated the FLS gene from roses, Petunia and peaches, and separated DFR from roses and Petunia. Gene. The results of tobacco transgenic show that FLS can enhance the synthesis of flavonol, reduce the accumulation of anthocyanin and make the color of transgenic tobacco change from red to white. On the contrary, overexpressing DFR gene, inhibiting the expression of endogenous Nt FLS and promoting the synthesis of anthocyanin. Based on the above results, we think that the competition of FLS and DFR to common substrates is divided. Do not achieve the synthesis of flavonols and anthocyanins, thus determining the differentiation of red and white flower color.2 using FPNI-PCR technology to separate Rr ANR from roses and construct a super expression vector to convert tobacco. The color of the transgenic tobacco is white, the content of proanthocyanidins and ABA is increased, and the antioxidant capacity is enhanced. Rr ANR transgenic tobacco and untransgenic tobacco are used. The results of the transcriptional sequence analysis showed that 1586 genes were up-regulated in transgenic tobacco, including related genes and related genes of secondary metabolites, especially the ABA pathway and antioxidant enzyme system. In order to clarify the molecular mechanism of Rr ANR to improve plant stress resistance, the Nt ABF. overexpression N was isolated from tobacco. T ABF significantly increased plant stress resistance and expression of antioxidant enzymes related genes. Exogenous PA and ABA treatments could also increase the expression of Nt ABF and antioxidant enzymes related genes. The above results showed that the overexpression of Rr ANR increased plant resistance by scavenging of reactive oxygen species and ABA signal.

【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S685.12

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