布渣叶转录组测序及ACGs生物合成关键基因的挖掘

发布时间：2018-01-12 19:37

本文关键词：布渣叶转录组测序及ACGs生物合成关键基因的挖掘　出处：《广东药科大学》2017年硕士论文　论文类型：学位论文

【摘要】：布渣叶(Microcos paniculata)是椴树科家族中的重要一员,其干燥叶片常被用作中药或“凉茶”原料。研究表明,布渣叶富含黄酮类成分。其中,芹菜素C-苷(Apigenin C-glycosides,ACGs)类代谢物含量丰富,具有抗炎、抗氧化、抗癌等多种生物活性。由于布渣叶资源逐步受到破坏,ACGs分离提取困难,致使ACGs的相关研究及开发利用较之其他黄酮类成分相对滞后。本文旨在以高通量转录组测序技术研究S1、S2、S3及S4四个不同生长阶段的布渣叶转录组,结合HPLC活性成分定量分析,以期挖掘布渣叶ACGs生物合成的关键基因,为布渣叶的种质保育、基因功能验证、时空表达调控机制等分子研究提供坚实的基础。具体实验内容及结果如下:(1)采集广东、广西、海南及云南四省共14个野生居群的布渣叶,提取其基因组DNA,扩增其ITS2和psbA-trnH序列。测序后进行多序列比对,结果显示布渣叶样品的ITS2序列存在12个变异位点,Pairwise Distance在0.000~0.020间;除S14(云南景洪)居群的样品在247bp~254bp处缺失一个8bp片段外,所有布渣叶样品的psbAtrnH序列完全一致,Pairwise Distance为0.000。基于布渣叶ITS2和psbA-trnH序列及其近源种植物相应条形码序列所构建的系统进化树显示14个居群的布渣叶样品均区别于其他近源种,聚为一个独立的单枝。综上,建立了以psbA-trnH为主,ITS2为辅的布渣叶DNA条形码鉴定体系。(2)提取S1、S2、S3及S4四个不同生长阶段布渣叶的总RNA,富集纯化mRNA,构建cDNA文库,采用Illumina Hiseq TM4000高通量测序平台进行转录组测序。Clean reads经de novo组装得到46,135条Unigene,平均长度为1,156bp,N50为1,946bp。在所有的Unigene中,有25,587(55.46%)条经BLAST在公共数据库Nr(30,085条),SwissProt(22,803条),KOG(18,761条)和KEGG(11,864条)中检索到高度同源序列而获得注释。以此布渣叶转录组数据库为基础,统计基因表达量,对差异基因进行GO富集分析、KEGG富集分析、趋势分析,以阐述布渣叶生长过程中基因表达模式的变化规律。(3)在转录组数据库注释的基础上,结合差异基因趋势分析与CDS结果预测,筛选得到ACGs生物合成相关的18个关键候选基因,分别为PAL(Unigene0032009、Unigene0032844),C4H(Unigene0032574),4CL(Unigene0026100、Unigene0029308),CHS(Unigene0016055、Unigene0018698),CHI(Unigene0016201),F2H(Unigene0021527),CGT(Unigene0014826、Unigene0026612),DHR(Unigene0020658),bHLH(Unigene0018327、Unigene0044249)和MYB(Unigene0017599、Unigene0030071、Unigene0044545、Unigene0045985)。RT-qPCR实验验证了候选基因在布渣叶生长过程中的表达模式与转录组所统计的结果相符,证明RNA-Seq定量基因的准确性。同时,采用HPLC测定S1、S2、S3及S4样品中的维采宁-1、维采宁-2、牡荆苷、异牡荆苷、夏佛塔苷、异夏佛塔苷、MpACG-2、MpACG-3及MpACG-4的相对含量。结果表明,大多数ACGs在布渣叶S1-S2生长阶段含量持续增加,在S2达到最大值,之后随着生长时间延长而含量逐渐减少,这与所筛选的候选基因的表达趋势十分相似。(4)通过ORF finder在线工具预测MpCHS2基因的开放阅读框,设计引物扩增其cDNA序列。产物与pET-30a(+)分别进行XhoI及BglII双酶切,在连接酶作用下构建MpCHS2-pET-30a(+)重组载体。将重组载体导入DH5α感受态细胞中,在含Kan抗生素(100μg/m L)LB固体培养基上培养,挑选阳性菌落,提取质粒进行Sanger测序。实验获得MpCHS2基因的序列,其cDNA长为1,176bp(GenBank登录号KY472608),编码391个氨基酸,预测其蛋白质分子量42.7KDa,理论等电点6.11,不含信号肽,不含跨膜域,所编码的蛋白质亲疏水性介于+2.422~-2.489间,具有两亲性。本论文结合了分子生物学、生物信息学、分析化学等多种学科技术,在建立布渣叶转录组信息库的基础上筛选了18个与ACGs生物合成相关的候选基因,并对其中的MpCHS2基因进行克隆,采用生物信息学分析、预测其编码产物的特征属性。本研究有助于从基因水平揭示布渣叶中ACGs的生物合成过程,为其代谢调控研究提供科学的依据,为ACGs的代谢工程储备一定的技术基础,更为布渣叶种质生物化学研究开阔新的研究思路。
[Abstract]:Microcospaniculata (Microcos paniculata) is an important member of the family Tiliaceae, the dry leaves are often used as traditional Chinese medicine or herbal tea raw materials. The results show that microcospaniculata is rich in flavonoids. Among them, apigenin glycosides (Apigenin C-glycosides, C- ACGs) metatolites content is rich, has anti-inflammatory, antioxidant. Antitumor. Because of microcospaniculata resources gradually damaged, difficult separation and extraction of ACGs, resulting in ACGs related research and development and utilization of other flavonoids than components is relatively backward. This paper aims to high-throughput transcriptome sequencing technology research S1, S2, S3 and S4 in four different growth stages of microcospaniculata transcriptome, combined with HPLC active ingredient quantitative analysis of key genes in order to excavate microcospaniculata ACGs biosynthesis, buzhaye for germplasm conservation, gene function, expression and molecular regulation mechanism research provide a solid foundation in detail. Check the contents and results are as follows: (1) collected from Guangdong, Guangxi, Hainan and Yunnan four provinces with a total of 14 wild populations of microcospaniculata group, extract its genomic DNA, amplification of the ITS2 and psbA-trnH sequences. After sequencing of multiple sequence alignment, the results showed that the ITS2 sequences of microcospaniculata samples there were 12 polymorphic sites, Pairwise Distance in 0.000~0.020; in addition to the S14 (Yunnan Jinghong) populations in the sample of 247bp~254bp deletion at a 8bp fragment, psbAtrnH sequence is completely consistent all microcospaniculata samples, Pairwise Distance 0.000. established microcospaniculata ITS2 and psbA-trnH sequences and near source species corresponding barcode sequence based on phylogenetic tree 14 populations of microcospaniculata samples are different from other kinds of near source, clustered into a single branch. In conclusion, based on the psbA-trnH based barcode identification system of cloth residue supplemented ITS2 leaves DNA. (2) to extract S1, S2, S3 and S4 four The total RNA in different growth stages of microcospaniculata, enrichment and purification of mRNA, a cDNA library was constructed using Illumina Hiseq TM4000 high-throughput sequencing platform transcriptome sequencing by de.Clean reads novo assembly of 46135 Unigene, the average length of 1156bp, N50 1946bp. in all Unigene, 25587 (55.46%) by BLAST the public database of Nr (30085), SwissProt (22803), KOG (18761) and KEGG (11864) to retrieve highly homologous sequences obtained by notes. Microcospaniculata transcriptome database as the foundation, the expression of GO gene in statistics, the difference of gene enrichment analysis, KEGG enrichment analysis, trend analysis in this process, the growth of microcospaniculata in change of gene expression pattern. (3) based on the transcriptome database annotation, combined with difference trend analysis and prediction results of CDS gene, screened 18 key ACGs biosynthesis Candidate genes were PAL (Unigene0032009, Unigene0032844), C4H (Unigene0032574), 4CL (Unigene0026100, Unigene0029308), CHS (Unigene0016055, Unigene0018698), CHI (Unigene0016201), F2H (Unigene0021527), CGT (Unigene0014826, Unigene0026612), DHR (Unigene0020658), bHLH (Unigene0018327, Unigene0044249 and MYB (Unigene0017599). Unigene0030071, Unigene0044545, Unigene0045985).RT-qPCR experiments verify the expression patterns of candidate genes and transcription group growth in microcospaniculata in the process of statistical results, prove the accuracy of RNA-Seq quantitative gene. At the same time, the determination of S1 by HPLC S2, S3 and S4 in the sample Weicai Ning -1, Weicai Ning -2, Vitexin. Isovitexin, Xia pagoda glycosides, isoschaftoside, MpACG-2, relative content of MpACG-3 and MpACG-4. The results show that most of the ACGs growth in microcospaniculata S1-S2 phase content increased, most reached at S2 Large value, after with the growth time and the content gradually reduced, the expression trend and candidate gene screening is very similar. (4) through the ORF finder online tools to predict the ORF of the MpCHS2 gene, amplified the cDNA sequence design primer. Products with pET-30a (+) respectively by XhoI and BglII digestion in the construction of MpCHS2-pET-30a, ligase (+) recombinant vector. The recombinant vector into alpha DH5 competent cells, the Kan containing antibiotics (100 g/m L) LB solid culture medium, selection of positive colonies, extraction of plasmid Sanger sequencing. The sequence of MpCHS2 gene, its length is 1176bp (cDNA GenBank accession number KY472608), encoding 391 amino acids, the predicted protein molecular weight is 42.7KDa, theoretical isoelectric point of 6.11, without signal peptide, without transmembrane domain, encoding protein hydrophobicity between +2.422~ -2.489, with two of the pro. The combination of molecular biology, bioinformatics, analytical chemistry and other disciplines, based on establishing microcospaniculata transcriptome information base on the screening of 18 candidate genes related to ACGs biosynthesis, and cloning of MpCHS2 gene which, by bioinformatic analysis, attribute prediction of its encoding product. The biosynthesis process of this research helps to reveal the ACGs microcospaniculata from the gene level, provide a scientific basis for the research of ACGs metabolic regulation, metabolic engineering, reserve a certain technical foundation for more research into the biochemistry of leaf germplasm cloth residue open new research ideas.

【学位授予单位】：广东药科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S567.19

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