本文選題：類黃酮 + 聯(lián)芐　； 參考：《山東大學(xué)》2017年碩士論文

【摘要】：苔類植物在植物進化史上具有特殊的地位,在適應(yīng)陸地生活的過程中產(chǎn)生了豐富的次級代謝產(chǎn)物,包括聯(lián)芐和黃酮、萜類、木脂素和香豆素等化合物。聯(lián)芐類化合物主要存在于苔類植物中,其生物活性多種多樣,涉及藥用價值范圍極廣,漸漸成為天然藥物研究以及新藥前導(dǎo)化合物開發(fā)的熱點。類黃酮的種類和功能多樣,對植物自身生長發(fā)育和人類健康都具有十分重要的意義。類黃酮的存在最早起源于苔蘚,在苔類植物中聯(lián)芐類化合物和類黃酮是共同存在的。類黃酮生物合成途徑起源于苯丙烷途徑,通過同位素標(biāo)記法證明聯(lián)芐類化合物的合成途徑也起源于苯丙烷途徑。在苔類植物中兩種化合物共存,共用其合成途徑的上游基因,包括與合成直接相關(guān)的結(jié)構(gòu)基因和調(diào)控合成途徑的轉(zhuǎn)錄因子。類黃酮生物合成途徑在高等植物中研究比較深入,許多關(guān)鍵的結(jié)構(gòu)基因和調(diào)控類黃酮合成的轉(zhuǎn)錄因子已經(jīng)被克隆和鑒定。在擬南芥中被歸為第Ⅲf亞族的bHLH轉(zhuǎn)錄因子是經(jīng)典的調(diào)控類黃酮合成的轉(zhuǎn)錄因子,如AtTT8。這類bHLH轉(zhuǎn)錄因子在其他植物中也多有報道,如葡萄中的VvMYC1、矮牽牛PhAN1、玉米ZmIN1等。本文從苔類植物鈍鱗紫背苔(Plagiochasma appendiculatum 和楔瓣地錢(Marchantia emarginata)的轉(zhuǎn)錄組測序數(shù)據(jù)中分別篩選出兩個注釋為bHLH家族的基因,經(jīng)序列分析發(fā)現(xiàn)兩個基因與AtTT8、VvMYC1同源性較高,具有典型的Basic-Helix1-Loop-Helix2結(jié)構(gòu)域,與Ⅲf亞族bHLH歸為一族,分別命名為PabHLH1和MebHLH1。通過3'-RACE獲得了 PabHLH1的基因全長序列信息,并成功地從鈍鱗紫背苔和楔瓣地錢的cDNA中克隆得到了 PabHLH1和MebHLH1的ORF序列。利用酵母單雜實驗證實PabHLH1和MebHLH1都具有轉(zhuǎn)錄激活區(qū),其轉(zhuǎn)錄活性區(qū)位于基因N-端。利用煙草瞬時表達研究基因亞細胞定位結(jié)果表明PabHLH1和MebHLH1定位于細胞核,表明其在細胞核中發(fā)揮其轉(zhuǎn)錄調(diào)節(jié)作用。將兩個基因分別構(gòu)建到植物表達載體pGWB5中,利用花序浸染法轉(zhuǎn)化擬南芥,使PabHLH1和MebHLH1在擬南芥中過表達。RT-PCR檢測PabHLH1和MebHLH1均成功的整合到擬南芥基因組中,且能正常的表達。PabHLH1在擬南芥中過表達可以使結(jié)構(gòu)基因PAL、CHS、CHI、F3H、DFR和FLS的表達水平上調(diào),類黃酮含量明顯增加,說明PabHLH1在擬南芥中能夠調(diào)控類黃酮化合物的合成。木質(zhì)素含量分析表明PabHLH1轉(zhuǎn)基因植株中木質(zhì)素含量與對照組沒有明顯的變化,但間苯三酚染色顯示MebHLH1可能上調(diào)了木質(zhì)素的合成。PabHLH1轉(zhuǎn)化鈍鱗紫背苔葉狀體,其基因轉(zhuǎn)錄水平與聯(lián)芐化合物成分的變化呈正比,表明PabHLH1可調(diào)控聯(lián)芐化合物的合成。苔類轉(zhuǎn)錄因子的功能鑒定對闡述高等植物分化前的生物合成途徑具有重要的生物學(xué)意義,也為提高天然產(chǎn)物活性成分提供了新的候選基因。黃酮類化合物結(jié)構(gòu)的多樣性也歸功于骨架形成后的結(jié)構(gòu)后修飾,如甲基化、糖基化以及�；揎椀�。黃酮的結(jié)構(gòu)修飾增加了黃酮化合物生理性能的多樣性,也改變了類黃酮的某些化學(xué)性質(zhì)。其中,甲氧基轉(zhuǎn)移酶(O-methyltransferases,OMTs)可以將黃酮化合物結(jié)構(gòu)上的羥基甲基化,增加了黃酮的疏水性,使黃酮類化合物的細胞膜穿透性增強,有利于腸道吸收及在體內(nèi)的結(jié)構(gòu)穩(wěn)定性。本文從鈍鱗紫背苔cDNA文庫中篩選并克隆得到了一個甲氧基轉(zhuǎn)移酶,命名為PaF6OMT。該酶具有鎂離子依賴性,屬于ClassⅠOMT,與小鼠兒茶酚OMT同源性比較高,在進化樹上與RnCatOMT聚為一簇。酶活結(jié)果表明PaF60MT的最適催化底物為野黃芩素和黃芩素,并能特異性的催化六位羥基分別生成千層紙素和高車前素,其酶活反應(yīng)條件簡單溫和,時間短,產(chǎn)物單一,催化產(chǎn)率高�？捎糜诿阜ê铣汕蛹埶睾透哕嚽八�。
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q943.2

【參考文獻】

相關(guān)期刊論文 前5條

1 侯銳;陳琦;王利;賈宇臣;李少偉;;花青素及其生物活性的研究進展[J];現(xiàn)代生物醫(yī)學(xué)進展;2015年28期

2 寇澤琪;閆德彪;馮鋒;;天然聯(lián)芐類化合物研究進展[J];海峽藥學(xué);2013年09期

3 郭英;瞿晶田;趙鑫;王虹;;天然產(chǎn)物千層紙素A的研究進展[J];遼寧中醫(yī)雜志;2012年12期

4 劉娜;婁紅祥;;雙聯(lián)芐類化合物結(jié)構(gòu)多樣性的研究進展[J];沈陽藥科大學(xué)學(xué)報;2011年08期

5 田維莉;孫守琴;;苔蘚植物生態(tài)功能研究新進展[J];生態(tài)學(xué)雜志;2011年06期

，

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