苔类植物两个bHLH转录因子和一个甲氧基转移酶的功能研究

发布时间：2018-06-06 01:54

本文选题：类黄酮 + 联苄　；参考：《山东大学》2017年硕士论文

【摘要】：苔类植物在植物进化史上具有特殊的地位,在适应陆地生活的过程中产生了丰富的次级代谢产物,包括联苄和黄酮、萜类、木脂素和香豆素等化合物。联苄类化合物主要存在于苔类植物中,其生物活性多种多样,涉及药用价值范围极广,渐渐成为天然药物研究以及新药前导化合物开发的热点。类黄酮的种类和功能多样,对植物自身生长发育和人类健康都具有十分重要的意义。类黄酮的存在最早起源于苔藓,在苔类植物中联苄类化合物和类黄酮是共同存在的。类黄酮生物合成途径起源于苯丙烷途径,通过同位素标记法证明联苄类化合物的合成途径也起源于苯丙烷途径。在苔类植物中两种化合物共存,共用其合成途径的上游基因,包括与合成直接相关的结构基因和调控合成途径的转录因子。类黄酮生物合成途径在高等植物中研究比较深入,许多关键的结构基因和调控类黄酮合成的转录因子已经被克隆和鉴定。在拟南芥中被归为第Ⅲf亚族的bHLH转录因子是经典的调控类黄酮合成的转录因子,如AtTT8。这类bHLH转录因子在其他植物中也多有报道,如葡萄中的VvMYC1、矮牵牛PhAN1、玉米ZmIN1等。本文从苔类植物钝鳞紫背苔(Plagiochasma appendiculatum 和楔瓣地钱(Marchantia emarginata)的转录组测序数据中分别筛选出两个注释为bHLH家族的基因,经序列分析发现两个基因与AtTT8、VvMYC1同源性较高,具有典型的Basic-Helix1-Loop-Helix2结构域,与Ⅲf亚族bHLH归为一族,分别命名为PabHLH1和MebHLH1。通过3'-RACE获得了 PabHLH1的基因全长序列信息,并成功地从钝鳞紫背苔和楔瓣地钱的cDNA中克隆得到了 PabHLH1和MebHLH1的ORF序列。利用酵母单杂实验证实PabHLH1和MebHLH1都具有转录激活区,其转录活性区位于基因N-端。利用烟草瞬时表达研究基因亚细胞定位结果表明PabHLH1和MebHLH1定位于细胞核,表明其在细胞核中发挥其转录调节作用。将两个基因分别构建到植物表达载体pGWB5中,利用花序浸染法转化拟南芥,使PabHLH1和MebHLH1在拟南芥中过表达。RT-PCR检测PabHLH1和MebHLH1均成功的整合到拟南芥基因组中,且能正常的表达。PabHLH1在拟南芥中过表达可以使结构基因PAL、CHS、CHI、F3H、DFR和FLS的表达水平上调,类黄酮含量明显增加,说明PabHLH1在拟南芥中能够调控类黄酮化合物的合成。木质素含量分析表明PabHLH1转基因植株中木质素含量与对照组没有明显的变化,但间苯三酚染色显示MebHLH1可能上调了木质素的合成。PabHLH1转化钝鳞紫背苔叶状体,其基因转录水平与联苄化合物成分的变化呈正比,表明PabHLH1可调控联苄化合物的合成。苔类转录因子的功能鉴定对阐述高等植物分化前的生物合成途径具有重要的生物学意义,也为提高天然产物活性成分提供了新的候选基因。黄酮类化合物结构的多样性也归功于骨架形成后的结构后修饰,如甲基化、糖基化以及酰基化修饰等。黄酮的结构修饰增加了黄酮化合物生理性能的多样性,也改变了类黄酮的某些化学性质。其中,甲氧基转移酶(O-methyltransferases,OMTs)可以将黄酮化合物结构上的羟基甲基化,增加了黄酮的疏水性,使黄酮类化合物的细胞膜穿透性增强,有利于肠道吸收及在体内的结构稳定性。本文从钝鳞紫背苔cDNA文库中筛选并克隆得到了一个甲氧基转移酶,命名为PaF6OMT。该酶具有镁离子依赖性,属于ClassⅠOMT,与小鼠儿茶酚OMT同源性比较高,在进化树上与RnCatOMT聚为一簇。酶活结果表明PaF60MT的最适催化底物为野黄芩素和黄芩素,并能特异性的催化六位羟基分别生成千层纸素和高车前素,其酶活反应条件简单温和,时间短,产物单一,催化产率高。可用于酶法合成千层纸素和高车前素。
[Abstract]:Moss has a special status in the history of plant evolution. It produces a rich secondary metabolite in the process of adapting to land life, including compounds such as benzyl and flavonoids, terpenes, lignans and coumarins. Bibenzyl compounds are mainly in the moss, and their biological activities are varied and involve a wide range of medicinal values. Gradually becoming a hot spot in the research of natural drugs and the development of new drug precursor compounds. The variety and function of flavonoids are of great significance to the growth and development of plants and human health. The existence of flavonoids was first originated from moss, and the benzyl compounds and flavonoids in the liverworts were common. The synthesis pathway originates in the phenylpropane pathway. The synthesis pathway of bibenzyl compounds is also derived from the phenylpropane pathway by isotope labeling. The two compounds in the moss plants coexist and share the upstream genes of the synthetic pathways, including the structural genes directly related to synthesis and the transcription factors regulating the synthetic pathway. The synthesis pathway is further studied in higher plants. Many key structural genes and transcription factors that regulate the synthesis of flavonoids have been cloned and identified. The bHLH transcription factor, which is classified as the third f subgroup in Arabidopsis, is a classic transcription factor that regulates the synthesis of flavonoids, such as AtTT8., bHLH transcription factors in other plants. There are many reports, such as VvMYC1 in grapes, Petunia PhAN1, corn ZmIN1 and so on. From the transcriptional sequence of Plagiochasma appendiculatum and Marchantia emarginata (Marchantia emarginata), two annotations are selected as bHLH family genes, and two genes are found to be homologous to AtTT8 and VvMYC1 by sequence analysis. It has a high sex and typical Basic-Helix1-Loop-Helix2 domain, which is classified as a family of bHLH of the third f subfamily. PabHLH1 and MebHLH1. are named PabHLH1 and MebHLH1. to obtain the whole sequence information of PabHLH1 gene, and the ORF sequence of PabHLH1 and MebHLH1 is successfully cloned from the cDNA of the blunt scale purple back moss and the wedge land money. The yeast single heterozygosity is used. The experiment confirmed that both PabHLH1 and MebHLH1 have a transcriptional activation area, and their transcriptional active regions are located at the N- end of the gene. The results of the subcellular localization of the transient expression of the gene in tobacco show that PabHLH1 and MebHLH1 are located in the nucleus, indicating that they play its transcriptional regulation in the nucleus. Two genes are constructed in the plant expression vector pGWB5, respectively. Arabidopsis thaliana was converted into Arabidopsis by inflorescence staining. The overexpression of PabHLH1 and MebHLH1 in Arabidopsis in Arabidopsis, PabHLH1 and MebHLH1 were successfully integrated into the Arabidopsis genome. The expression of.PabHLH1 in Arabidopsis could increase the expression level of PAL, CHS, CHI, F3H, DFR and FLS, and the flavonoid content was significantly increased. The results showed that PabHLH1 could regulate the synthesis of flavonoids in Arabidopsis. Lignin content analysis showed that the content of lignin in PabHLH1 transgenic plants did not change obviously with the control group, but the staining of benzene three phenol showed that MebHLH1 might increase the synthesis of lignin synthesis.PabHLH1 conversion blunt scale purple back moss, and its gene transcription water It is proportional to the changes in the composition of bibenzyl compounds, indicating that PabHLH1 can regulate the synthesis of bibenzyl compounds. The functional identification of the liverwort transcription factors has important biological significance for the biosynthesis pathway before the differentiation of higher plants, and a new candidate gene for improving the active components of natural products. The structure of flavonoids. Diversity is also attributed to post structural modification, such as methylation, glycosylation, and acylation modification. The structural modification of flavonoids increases the physiological diversity of flavonoids and also changes some chemical properties of flavonoids. Among them, O-methyltransferases (OMTs) can structure flavonoids. The hydroxy methylation on the upper side increases the hydrophobicity of flavonoids, enhances the penetration of the cell membrane of the flavonoids, is beneficial to the absorption of the intestine and the stability of the structure in the body. A methoxy transferase is screened and cloned from the cDNA Library of the blunt scale purple back moss, named PaF6OMT., which is of magnesium ion dependence and belongs to the Class I. OMT, which has high homology with the mouse catechol OMT, is clustered with RnCatOMT in the evolutionary tree. The results of enzyme activity show that the most suitable catalytic substrates for PaF60MT are Scutellaria and baicalein, and the six hydroxyl groups can be specifically catalyzed to produce 1000 layers of paper and high precursor respectively. The enzyme activity is simple and mild, the time is short, the product is single, and the product is catalyzed. It can be used for enzymatic synthesis of Melaleuca and high Plantin.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2

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