蓝色花种质筛选及其花瓣片类黄酮化合物分析研究

发布时间：2018-03-05 02:32

本文选题：蓝色花　切入点：华丽龙胆　出处：《昆明理工大学》2017年硕士论文　论文类型：学位论文

【摘要】：本研究结合植物化学和分子生物学的方法对三种蓝色花的类黄酮化合物进行分析,推定花青素苷代谢途径,对华丽龙胆三个开花阶段进行转录组测序,挖掘差异表达基因并分析其表达模式。1、使用色差仪对三种蓝色花植物的花瓣进行表型测定,使用高效液相色谱法(HPLC-PAD)对花青素苷含量和中间代谢产物进行测定,使用高效液质联用法(HPLC-ESI-MS)对花青素苷的成分进行测定。得到的结果如下:(1)蓝亚麻的b*值为-23.9,花青素苷成分为飞燕草素苷、矢车菊素苷和锦葵素苷,三种花青素苷均得到了酰基化修饰,其中飞燕草素苷占比例为60%;(2)蓝玉簪龙胆的b*值为-22.1,花青素苷成分为天竺葵素苷、矢车菊素苷和飞燕草素苷,其中只有飞燕草素苷获得了酰基化修饰;(3)华丽龙胆的b*值为-36.82,花青素苷成分为飞燕草素苷和天竺葵素苷,并且均得到了酰基化修饰,飞燕草素苷含量占95%以上。综合分析三种蓝色花的类黄酮化合物,推定了三种蓝色花植物的类黄酮代谢途径。其中,华丽龙胆的8种花青素苷都得到了酰基化修饰,飞燕草素苷的比例占总花青素苷的95%以上,最终选择华丽龙胆进行下一步研究。2、经过表型和色素的比较分析,选择华丽龙胆进行转录组测序,对华丽龙胆的三个开花阶段提取总RNA后,使用Illumina HiSeqTM2000的平台进行转录组测序。对转录组的测序结果进行组装,基因功能注释等,构建转录组数据库。在Nr数据库中得到注释的Unigene有39169条,在SwissProt数据库中得到注释的Unigene有27212条,在KEGG数据库中得到注释的Unigene有23418条,在COG/KOG数据库中得到注释的Unigene 有 10649 条。根据华丽龙胆三个开花阶段的基因表达统计差异基因,H1-R到H2-R阶段显著上调的基因有8995个,显著下调的基因有20121个;H2-R到H5-R阶段显著上调的基因有12800个,显著下调的基因有13439个;H1-R到H5-R阶段显著上调的基因有8097个,显著下调的基因有19699个。根据Unigene功能注释,筛选到花色相关的基因共有127条。3、本研究对三个开花阶段的华丽龙胆进行基因表达量验证,对9个Unigene(Unigene0037993 功能注释为 CHS、Unigene0023341 功能注释为 FLS、Unigene0014078功能注释为F3'5'H、Unigene0055940功能注释为DFR、Unigene0059995功能注释为3GT、Unigene0053146 功能注释为 3'GT、Unigene0054587 功能注释为 3,5GT、Unigene0051274功能注释为5AT、Unigene0012123功能注释为GST)进行荧光定量PCR验证,结果发现,荧光定量PCR结果与转录组测序表达量的变化幅度一致,证明转录组测序结果可靠。4、对表达量验证的9个基因进行表达模式分析,Unigene0037993、Unigene0014078、Unigene0055940、Unigene0054587、Unigene0051274 和 Unigene0012123 这 6 个基因的表达模式呈现先升高后降低的趋势,与花青素苷含量的变化趋势相近,推测与花青素苷合成相关。其中,注释为FLS和DFR的两个基因的表达量变化趋势相反,共享相同的底物,呈现竞争的关系。
[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.
【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2;Q946

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