基于RNA-Seq的粘毛黄芩黄酮类化合物合成基因及转录因子的表达研究

发布时间：2018-05-11 03:14

本文选题：粘毛黄芩 + 转录组测序　；参考：《陕西师范大学》2016年硕士论文

【摘要】：粘毛黄芩(Scutellaria viscidula Bunge)为唇形科(Labiatae)黄芩属(Scutellaria)多年生草本植物,以干燥根入药,主要活性成分为黄酮类化合物(如黄芩苷、黄芩素等),具有抗心脑血管疾病、抗癌、抗炎等多种生物活性。目前,药用植物基因组数据相对匮乏,遗传背景及其基因信息严重不足,导致大多数药用植物活性成分合成的途径及分子机制不清楚。随着转录组测序技术的快速发展和完善,利用转录组测序获得的大量基因信息数据揭示药用植物次生代谢生物合成途径及其关键酶基因克隆、表达调控等分子机制成为药用植物研究热点。目前,国内对粘毛黄芩的研究主要集中在资源评价利用和部分植物表达载体的构建等,尚不清楚药用活性成分黄酮类化合物(如黄芩苷、黄芩素等)合成的遗传基础,更缺少合成途径中关键酶基因结构及表达调控等相关信息。本研究以粘毛黄芩为材料,通过转录组测序构建转录组数据库,在此基础上挖掘黄酮合成途径关键基因,并通过荧光定量PCR分析了结构基因和调控转录因子的表达,结合总黄酮积累动态变化和基因表达,初步探讨粘毛黄芩药用活性成分类黄酮的合成途径及其分子机制。本研究主要获得以下研究结果：1.以粘毛黄芩为材料,提取RNA,基于Illumina HiSeq/MiSeq 进行转录组测序,共获得reads数42310834条,组装出40052个Unigenes, Unigenes平均读长882bp,N50读长1577bp。将粘毛黄芩测序获得的Unigenes同6个蛋白数据库(NR、KO、SwissProt、Pfam、GO、KOG)和Nt核酸数据库进行Blast比对,共有24892条(62.14%)序列得到注释。通过与KOG数据库的比对,粘毛黄芩基因被分到26个不同的功能组中,通过Blast比对和ESTScan软件预测,共获得33367条CDS记录。在此基础上建立粘毛黄芩转录组数据库,为粘毛黄芩基因工程育种和活性成分形成分子机制探索提出新的研究思路和方法。2.通过对数据库中SSR进行统计分析,粘毛黄芩共检测出8925个SSRs位点,其中二核苷酸是主要的重复类型(占总SSRs的51.69%),其次是单、三核苷酸重复(29.03%,18.32%)。SSRs位点中优势二核苷酸基元类型是AG/CT(占总SSRs的40.78%)。SSR信息分析将为粘毛黄芩功能基因的挖掘和分子标记辅助选择育种等奠定了基础。3.对粘毛黄芩黄酮类化合物合成相关基因代谢通路进行生物信息学分析,结果有136条注释到苯丙烷代谢途径,41条注释到黄酮类合成途径。其中注释获得有4条查尔酮合酶(CHS)、1条查尔酮异构酶(CHI)、2条黄烷酮3-羟化酶(F3H)、11条苯丙氨酸解氨酶(PAL)、5条4-香豆酰辅酶A连接酶(4CL)基因。另外,在粘毛黄芩转录组数据库中还发现与黄酮类化合物合成相关MYB转录因子序列(MYB)88条,碱性螺旋-环-螺旋转录因子序列(bHLH)34条和WD40重复蛋白转录因子序列(WD40)10条。4.通过NCBI数据库和生物信息学分析,筛选和鉴定出粘毛黄芩黄酮类化合物生物合成相关基因(CHI、CHS和F3H)和转录因子(bHLH, MYB2)的部分序列,并以此为模板设计引物。应用荧光定量PCR技术对上述粘毛黄芩5个黄酮类化合物生物合成相关基因在三个时期的根、茎和叶的表达量进行检测,结果表明,三个时期根、茎和叶均检测到基因不同程度表达,CHS、F3H和CHI在7月份各部位表达量较高。另外,MYB2基因在三个时期的表达量呈现下降趋势,表现出同一部位中的表达可能影响CHS和CHI的表达,暗示对CHS的正向调控,而对CHI的负向调控,但不具显著相关性。5.提取粘毛黄芩三个时期不同部位的总黄酮,以黄芩苷为标准品,利用紫外分光光度法测定根、茎和叶在三个不同时期总黄酮含量,结果发现7月份三个部位总黄酮含量显著高于5月和9月,且均呈现出根茎叶。结合各时期CHS、F3H和CHI的表达量,初步显示在7月份总黄酮含量较高时,CHS、F3H和CHI基因的表达量也较高,暗示基因表达与总黄酮含量间可能存在正相关。上述研究获得的粘毛黄芩转录组数据、黄酮合成途径关键基因和转录因子,初步探讨了粘毛黄芩黄酮类化合物合成途径关键基因表达与总黄酮量的关系,为进一步揭示粘毛黄芩活性成分形成分子机制和通过基因工程手段提高药用成分含量提供依据,具有重要的理论和实际意义。
[Abstract]:Scutellaria viscidula Bunge (Scutellaria baicalensis) is a perennial herbaceous plant of Huang Qinshu (Scutellaria) in the family of lip family (Labiatae). The main active ingredients are flavonoids (baicalin, baicalein, etc.) with dry roots, which have many biological activities, such as anti cardio cerebrovascular disease, anticancer, anti-inflammatory and so on. Lack of genetic background and genetic information, which leads to the inadequacy of the pathway and molecular mechanism for the synthesis of active ingredients in most medicinal plants. With the rapid development and improvement of the sequencing technology, a large number of gene information data obtained from the sequence of transcriptional sequences are used to reveal the secondary metabolic biosynthesis pathway and the key enzyme base of the medicinal plants. Molecular mechanisms such as cloning, expression and regulation have become a hot topic in the research of medicinal plants. At present, the research on Scutellaria baicalensis is mainly concentrated on the utilization of resources and the construction of some plant expression vectors. It is not clear that the genetic basis for the synthesis of flavonoids such as baicalin, baicalein and so on is not clear. The key enzyme gene structure and expression regulation and other related information. This study uses Scutellaria baicalensis as material to construct a transcriptional database by sequencing the transcriptional group. On this basis, the key genes of the flavonoid synthesis pathway are excavated, and the expression of structural genes and regulatory transcripts is analyzed by fluorescence quantitative PCR, and the accumulation of dynamic changes and bases of total flavonoids is combined with the total flavonoids. The synthesis pathway and molecular mechanism of the medicinal active ingredients of Scutellaria baicalensis were preliminarily discussed. The main results were as follows: 1. RNA was extracted from Scutellaria baicalensis Georgi and Illumina HiSeq/MiSeq was sequenced based on Illumina HiSeq/MiSeq. The total number of reads was obtained, and 40052 Unigenes and Unigenes average reading length was assembled. 882bp, N50 read long 1577bp. to compare Unigenes with 6 protein databases (NR, KO, SwissProt, Pfam, GO, KOG) and Nt nucleic acid database of Scutellaria baicalensis. A total of 24892 (62.14%) sequences were annotated. A total of 33367 CDS records were obtained by can software. On this basis, the database of Scutellaria baicalensis transcriptional group was set up to provide new research ideas and methods for exploring the molecular mechanism of genetic engineering breeding and active component formation of Scutellaria baicalensis Georgi,.2. through the statistical analysis of SSR in the database, 8925 SSRs sites were detected in the Scutellaria baicalensis Georgi, of which two Nucleotides are the main repeat types (51.69% of the total SSRs), followed by single, trinucleotide repeat (29.03%, 18.32%).SSRs sites, the dominant dinucleotide primitives are AG/CT (accounting for 40.78% of the total SSRs).SSR information analysis will provide a basis for the mining of Scutellaria baicalensis functional gene and sub marker assisted selection breeding, and so on, which lays the foundation of.3. for Scutellaria baicalensis. Bioinformatics analysis of the metabolic pathway of the flavonoid synthesis related genes was carried out. The results were 136 annotated to the phenylpropane metabolic pathway and 41 annotated to the flavonoid synthesis pathway. Among them, there were 4 chalcone synthase (CHS), 1 chalcone isomerase (CHI), 2 xanone 3- hydroxylase (F3H), 11 phenylalanine ammonia lyase (PAL), 5 4. - coumaryl coenzyme A ligase (4CL) gene. In addition, in the database of Scutellaria baicalensis transcriptional group, the MYB transcription factor sequence (MYB) 88, 34 basic spiral loop helix transcription factor sequence (bHLH) and WD40 repeat protein transcriptional factor sequence (WD40) 10.4. through NCBI database and bioinformatics credits Analysis, screening and identification of the partial sequences of CHI, CHS and F3H and bHLH (MYB2) related genes of flavonoids of Scutellaria baicalensis Scutellaria, and designed the primers as a template. The expression of the related genes of 5 flavonoids of Scutellaria baicalensis Scutellaria by fluorescence quantitative PCR technique was used to express the expression of the root, stem and leaf in the three periods. The results showed that the expression of CHS, F3H and CHI was higher in the three stages, and the expression of MYB2 gene in the three periods was lower in July, and the expression in the same site may affect the expression of CHS and CHI, suggesting the positive regulation of CHS. Negative regulation of CHI, but without significant correlation.5. extraction of total flavonoids from three different parts of Scutellaria baicalensis, using baicalin as the standard, using UV spectrophotometry to determine the total flavonoids content of root, stem and leaf at three different periods. The results showed that the total flavonoids content in three parts of July was significantly higher than that in May and September. The expression of CHS, F3H and CHI in each period showed that the expression of CHS, F3H and CHI genes was also higher in July, suggesting that there might be a positive correlation between the gene expression and the total flavonoids content. The relationship between the expression of key genes in the synthetic pathway of flavonoids in Scutellaria baicalensis and the amount of total flavonoids is discussed. It is of great theoretical and practical significance to further reveal the molecular mechanism of the active components of Scutellaria baicalensis and to improve the content of medicinal ingredients through genetic engineering.

【学位授予单位】：陕西师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2;S567.239

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