本文選題：藍色花　切入點：華麗龍膽　出處：《昆明理工大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：本研究結(jié)合植物化學(xué)和分子生物學(xué)的方法對三種藍色花的類黃酮化合物進行分析,推定花青素苷代謝途徑,對華麗龍膽三個開花階段進行轉(zhuǎn)錄組測序,挖掘差異表達基因并分析其表達模式。1、使用色差儀對三種藍色花植物的花瓣進行表型測定,使用高效液相色譜法(HPLC-PAD)對花青素苷含量和中間代謝產(chǎn)物進行測定,使用高效液質(zhì)聯(lián)用法(HPLC-ESI-MS)對花青素苷的成分進行測定。得到的結(jié)果如下:(1)藍亞麻的b*值為-23.9,花青素苷成分為飛燕草素苷、矢車菊素苷和錦葵素苷,三種花青素苷均得到了�；揎�,其中飛燕草素苷占比例為60%;(2)藍玉簪龍膽的b*值為-22.1,花青素苷成分為天竺葵素苷、矢車菊素苷和飛燕草素苷,其中只有飛燕草素苷獲得了�；揎�;(3)華麗龍膽的b*值為-36.82,花青素苷成分為飛燕草素苷和天竺葵素苷,并且均得到了�；揎�,飛燕草素苷含量占95%以上。綜合分析三種藍色花的類黃酮化合物,推定了三種藍色花植物的類黃酮代謝途徑。其中,華麗龍膽的8種花青素苷都得到了�；揎�,飛燕草素苷的比例占總花青素苷的95%以上,最終選擇華麗龍膽進行下一步研究。2、經(jīng)過表型和色素的比較分析,選擇華麗龍膽進行轉(zhuǎn)錄組測序,對華麗龍膽的三個開花階段提取總RNA后,使用Illumina HiSeqTM2000的平臺進行轉(zhuǎn)錄組測序。對轉(zhuǎn)錄組的測序結(jié)果進行組裝,基因功能注釋等,構(gòu)建轉(zhuǎn)錄組數(shù)據(jù)庫。在Nr數(shù)據(jù)庫中得到注釋的Unigene有39169條,在SwissProt數(shù)據(jù)庫中得到注釋的Unigene有27212條,在KEGG數(shù)據(jù)庫中得到注釋的Unigene有23418條,在COG/KOG數(shù)據(jù)庫中得到注釋的Unigene 有 10649 條。根據(jù)華麗龍膽三個開花階段的基因表達統(tǒng)計差異基因,H1-R到H2-R階段顯著上調(diào)的基因有8995個,顯著下調(diào)的基因有20121個;H2-R到H5-R階段顯著上調(diào)的基因有12800個,顯著下調(diào)的基因有13439個;H1-R到H5-R階段顯著上調(diào)的基因有8097個,顯著下調(diào)的基因有19699個。根據(jù)Unigene功能注釋,篩選到花色相關(guān)的基因共有127條。3、本研究對三個開花階段的華麗龍膽進行基因表達量驗證,對9個Unigene(Unigene0037993 功能注釋為 CHS、Unigene0023341 功能注釋為 FLS、Unigene0014078功能注釋為F3'5'H、Unigene0055940功能注釋為DFR、Unigene0059995功能注釋為3GT、Unigene0053146 功能注釋為 3'GT、Unigene0054587 功能注釋為 3,5GT、Unigene0051274功能注釋為5AT、Unigene0012123功能注釋為GST)進行熒光定量PCR驗證,結(jié)果發(fā)現(xiàn),熒光定量PCR結(jié)果與轉(zhuǎn)錄組測序表達量的變化幅度一致,證明轉(zhuǎn)錄組測序結(jié)果可靠。4、對表達量驗證的9個基因進行表達模式分析,Unigene0037993、Unigene0014078、Unigene0055940、Unigene0054587、Unigene0051274 和 Unigene0012123 這 6 個基因的表達模式呈現(xiàn)先升高后降低的趨勢,與花青素苷含量的變化趨勢相近,推測與花青素苷合成相關(guān)。其中,注釋為FLS和DFR的兩個基因的表達量變化趨勢相反,共享相同的底物,呈現(xiàn)競爭的關(guān)系。
[Abstract]:In this study, phytochemistry and molecular biology were used to analyze the flavonoids of three blue flowers and to deduce the pathway of anthocyanin metabolism, and to sequence the transcriptome of three flowering stages of Gentiana gentiana. The differentially expressed genes were excavated and their expression patterns were analyzed. The phenotypic characteristics of the petals of three blue flower plants were determined by colorimetry, and the anthocyanin content and intermediate metabolites were determined by high performance liquid chromatography (HPLC). The composition of anthocyanin was determined by HPLC-ESI-MS. the results were as follows: (1) the value of b * was -23.9. the anthocyanin was composed of swallow oxaloside, cornulin and melamine, and the content of anthocyanin was determined by HPLC-ESI-MS.The results showed that the content of anthocyanin was -23.9. All three anthocyanins were acylated, among them, the ratio of swallows glycoside was 60) the value of b * of Gentiana oleracea was -22.1, and the components of anthocyanin were geranium glucoside, cornulin glycoside and verdanin glycoside. Among them, only the acylated modified gentian was obtained by acylation modification. The b * value of the gentian was -36.82, and the anthocyanin was composed of the swallows and geranium glucosides, and both of them were acylated. The flavonoid compounds of three blue flowers were analyzed synthetically, and the flavonoid metabolic pathways of three blue flower plants were deduced. Among them, 8 anthocyanins of Gentiana gentianica were modified by acylation. More than 95%% of total anthocyanin was accounted for by swallows. The next step was to choose Gentiana gentiana for further study. After comparative analysis of phenotypes and pigments, Gentianopsis gentianum was selected for transcriptional sequencing. After extracting total RNA from the three flowering stages of Gentiana gentiana, the transcriptome sequencing was carried out using the platform of Illumina HiSeqTM2000. The sequencing results of the transcriptional groups were assembled, the gene function was annotated, and so on. There are 39169 Unigene annotated in Nr database, 27212 Unigene annotated in SwissProt database, 23418 Unigene annotated in KEGG database. There were 10649 annotated Unigene in COG/KOG database. According to the statistical difference of gene expression in three flowering stages of Gentiana gentiana, 8995 genes were significantly up-regulated in H1-R to H2-R stages. There were 12800 genes significantly up-regulated in the H2-R to H5-R stages, 13439 genes significantly up-regulated in H1-R to H5-R stages, 8097 genes significantly up-regulated and 19699 genes significantly down-regulated. According to the Unigene functional annotation, A total of 127 genes related to flower color were screened. In this study, the gene expression of three flowering stages of Gentiana gentiana was verified. Fluorescence quantitative PCR verification was performed on 9 Unigene(Unigene0037993 functional annotations named CHSU Unigene0023341, FLSU Unigene0014078, F3GN, Unigene0055940, DFR Unigene0059995, DFRG Unigene0053146, 3GTU Unigene0054587, 5ATT Unigene0051274, 5ATT Unigene0012123, GSTs. The results of fluorescent quantitative PCR were consistent with the changes of transcriptome sequencing expression. The results showed that the results of transcriptome sequencing were reliable. 4. The expression patterns of 9 genes verified by transcriptome sequencing were analyzed. The expression patterns of the six genes, Unigene0037993, Unigene0014078, Unigene0055940, Unigene0054587, Unigene0051274 and Unigene0012123, showed a trend of first increasing and then decreasing, similar to that of anthocyanin content. The expression level of the two genes annotated as FLS and DFR showed the opposite trend, sharing the same substrate and showing a competitive relationship.
【學(xué)位授予單位】：昆明理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q943.2;Q946

【參考文獻】

相關(guān)期刊論文 前10條

1 楊少勇,樊國盛;云南省藍色花植物研究初探[J];西南林學(xué)院學(xué)報;2002年03期

2 楊少勇,安銀嶺,樊國盛,畢望富,王沙生;藍色花植物花色素的著色機理[J];北京林業(yè)大學(xué)學(xué)報;2003年05期

3 柴長宏;天藍龍膽的栽培技術(shù)[J];中國林副特產(chǎn);2004年03期

4 張明麗;胡永紅;秦俊;;城市植物群落的減噪效果分析[J];植物資源與環(huán)境學(xué)報;2006年02期

5 李莉;孫欣;馬君蘭;趙越;;異黃酮合成代謝調(diào)控關(guān)鍵酶CHS、CHI的特性與研究前景[J];大豆科學(xué);2007年05期

6 李海峰;;天藍龍膽草的試管開花及花后管理[J];中國花卉園藝;2007年06期

7 李義龍;肇濤瀾;陳立超;趙亞茹;權(quán)太勇;;花色素苷生物合成及花色的調(diào)控[J];生命科學(xué);2008年01期

8 李崇暉;王亮生;舒慶艷;徐彥軍;張潔;;迎紅杜鵑花色素組成及花色在開花過程中的變化[J];園藝學(xué)報;2008年07期

9 張劍亮;潘大仁;周以飛;王占成;華樹妹;侯黎麗;隨粉粉;;觀賞向日葵花青素苷合成途徑同源基因的克隆與表達[J];園藝學(xué)報;2009年01期

10 侯夫云;王慶美;李愛賢;張海燕;董順旭;解備濤;;植物花青素合成酶的研究進展[J];中國農(nóng)學(xué)通報;2009年21期

相關(guān)博士學(xué)位論文 前3條

1 金雪花;基于高通量測序的瓜葉菊花青素苷合成途徑研究[D];北京林業(yè)大學(xué);2013年

2 胡可;花青素苷合成途徑中結(jié)構(gòu)基因的表達對菊花和瓜葉菊花色的影響[D];北京林業(yè)大學(xué);2010年

3 孟麗;藍色花形成關(guān)鍵基因的分離及其表達分析[D];北京林業(yè)大學(xué);2006年

相關(guān)碩士學(xué)位論文 前2條

1 熊梅;蠅蛆轉(zhuǎn)錄組學(xué)分析及Zn~(2+)對其金屬硫蛋白基因表達的影響[D];華中農(nóng)業(yè)大學(xué);2015年

2 裴仁濟;非洲紫羅蘭藍色花顯色機制研究[D];天津農(nóng)學(xué)院;2011年

，

本文編號：1568390