本文關(guān)鍵詞：布渣葉轉(zhuǎn)錄組測(cè)序及ACGs生物合成關(guān)鍵基因的挖掘　出處：《廣東藥科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 布渣葉 DNA條形碼 轉(zhuǎn)錄組 ACGs 候選基因

【摘要】：布渣葉(Microcos paniculata)是椴樹科家族中的重要一員,其干燥葉片常被用作中藥或“涼茶”原料。研究表明,布渣葉富含黃酮類成分。其中,芹菜素C-苷(Apigenin C-glycosides,ACGs)類代謝物含量豐富,具有抗炎、抗氧化、抗癌等多種生物活性。由于布渣葉資源逐步受到破壞,ACGs分離提取困難,致使ACGs的相關(guān)研究及開發(fā)利用較之其他黃酮類成分相對(duì)滯后。本文旨在以高通量轉(zhuǎn)錄組測(cè)序技術(shù)研究S1、S2、S3及S4四個(gè)不同生長(zhǎng)階段的布渣葉轉(zhuǎn)錄組,結(jié)合HPLC活性成分定量分析,以期挖掘布渣葉ACGs生物合成的關(guān)鍵基因,為布渣葉的種質(zhì)保育、基因功能驗(yàn)證、時(shí)空表達(dá)調(diào)控機(jī)制等分子研究提供堅(jiān)實(shí)的基礎(chǔ)。具體實(shí)驗(yàn)內(nèi)容及結(jié)果如下:(1)采集廣東、廣西、海南及云南四省共14個(gè)野生居群的布渣葉,提取其基因組DNA,擴(kuò)增其ITS2和psbA-trnH序列。測(cè)序后進(jìn)行多序列比對(duì),結(jié)果顯示布渣葉樣品的ITS2序列存在12個(gè)變異位點(diǎn),Pairwise Distance在0.000~0.020間;除S14(云南景洪)居群的樣品在247bp~254bp處缺失一個(gè)8bp片段外,所有布渣葉樣品的psbAtrnH序列完全一致,Pairwise Distance為0.000�；诓荚~ITS2和psbA-trnH序列及其近源種植物相應(yīng)條形碼序列所構(gòu)建的系統(tǒng)進(jìn)化樹顯示14個(gè)居群的布渣葉樣品均區(qū)別于其他近源種,聚為一個(gè)獨(dú)立的單枝。綜上,建立了以psbA-trnH為主,ITS2為輔的布渣葉DNA條形碼鑒定體系。(2)提取S1、S2、S3及S4四個(gè)不同生長(zhǎng)階段布渣葉的總RNA,富集純化mRNA,構(gòu)建cDNA文庫(kù),采用Illumina Hiseq TM4000高通量測(cè)序平臺(tái)進(jìn)行轉(zhuǎn)錄組測(cè)序。Clean reads經(jīng)de novo組裝得到46,135條Unigene,平均長(zhǎng)度為1,156bp,N50為1,946bp。在所有的Unigene中,有25,587(55.46%)條經(jīng)BLAST在公共數(shù)據(jù)庫(kù)Nr(30,085條),SwissProt(22,803條),KOG(18,761條)和KEGG(11,864條)中檢索到高度同源序列而獲得注釋。以此布渣葉轉(zhuǎn)錄組數(shù)據(jù)庫(kù)為基礎(chǔ),統(tǒng)計(jì)基因表達(dá)量,對(duì)差異基因進(jìn)行GO富集分析、KEGG富集分析、趨勢(shì)分析,以闡述布渣葉生長(zhǎng)過程中基因表達(dá)模式的變化規(guī)律。(3)在轉(zhuǎn)錄組數(shù)據(jù)庫(kù)注釋的基礎(chǔ)上,結(jié)合差異基因趨勢(shì)分析與CDS結(jié)果預(yù)測(cè),篩選得到ACGs生物合成相關(guān)的18個(gè)關(guān)鍵候選基因,分別為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實(shí)驗(yàn)驗(yàn)證了候選基因在布渣葉生長(zhǎng)過程中的表達(dá)模式與轉(zhuǎn)錄組所統(tǒng)計(jì)的結(jié)果相符,證明RNA-Seq定量基因的準(zhǔn)確性。同時(shí),采用HPLC測(cè)定S1、S2、S3及S4樣品中的維采寧-1、維采寧-2、牡荊苷、異牡荊苷、夏佛塔苷、異夏佛塔苷、MpACG-2、MpACG-3及MpACG-4的相對(duì)含量。結(jié)果表明,大多數(shù)ACGs在布渣葉S1-S2生長(zhǎng)階段含量持續(xù)增加,在S2達(dá)到最大值,之后隨著生長(zhǎng)時(shí)間延長(zhǎng)而含量逐漸減少,這與所篩選的候選基因的表達(dá)趨勢(shì)十分相似。(4)通過ORF finder在線工具預(yù)測(cè)MpCHS2基因的開放閱讀框,設(shè)計(jì)引物擴(kuò)增其cDNA序列。產(chǎn)物與pET-30a(+)分別進(jìn)行XhoI及BglII雙酶切,在連接酶作用下構(gòu)建MpCHS2-pET-30a(+)重組載體。將重組載體導(dǎo)入DH5α感受態(tài)細(xì)胞中,在含Kan抗生素(100μg/m L)LB固體培養(yǎng)基上培養(yǎng),挑選陽性菌落,提取質(zhì)粒進(jìn)行Sanger測(cè)序。實(shí)驗(yàn)獲得MpCHS2基因的序列,其cDNA長(zhǎng)為1,176bp(GenBank登錄號(hào)KY472608),編碼391個(gè)氨基酸,預(yù)測(cè)其蛋白質(zhì)分子量42.7KDa,理論等電點(diǎn)6.11,不含信號(hào)肽,不含跨膜域,所編碼的蛋白質(zhì)親疏水性介于+2.422~-2.489間,具有兩親性。本論文結(jié)合了分子生物學(xué)、生物信息學(xué)、分析化學(xué)等多種學(xué)科技術(shù),在建立布渣葉轉(zhuǎn)錄組信息庫(kù)的基礎(chǔ)上篩選了18個(gè)與ACGs生物合成相關(guān)的候選基因,并對(duì)其中的MpCHS2基因進(jìn)行克隆,采用生物信息學(xué)分析、預(yù)測(cè)其編碼產(chǎn)物的特征屬性。本研究有助于從基因水平揭示布渣葉中ACGs的生物合成過程,為其代謝調(diào)控研究提供科學(xué)的依據(jù),為ACGs的代謝工程儲(chǔ)備一定的技術(shù)基礎(chǔ),更為布渣葉種質(zhì)生物化學(xué)研究開闊新的研究思路。
[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.

【學(xué)位授予單位】：廣東藥科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S567.19

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 馮亮;羅文匯;;布渣葉總生物堿在高脂血癥大鼠中的作用機(jī)制分析[J];中國(guó)醫(yī)學(xué)創(chuàng)新;2016年17期

2 江潔怡;李養(yǎng)學(xué);李素梅;胥愛麗;;HPLC同時(shí)測(cè)定布渣葉總黃酮提取物中6種黃酮類成分[J];中國(guó)實(shí)驗(yàn)方劑學(xué)雜志;2016年09期

3 陳偉韜;梁之桃;周立敏;周勁松;趙中振;;布渣葉超高效液相指紋圖譜研究[J];世界中醫(yī)藥;2016年02期

4 李菁;向婷婷;許成;劉朝奇;張長(zhǎng)城;袁丁;周志勇;;分子譜系地理學(xué)在藥用植物基因水平的研究進(jìn)展[J];中草藥;2015年08期

5 王華;李茂福;楊媛;金萬梅;;果實(shí)花青素生物合成分子機(jī)制研究進(jìn)展[J];植物生理學(xué)報(bào);2015年01期

6 陳艷芬;楊超燕;李坤平;曾宇;王穎芳;陳美燕;;布渣葉總黃酮對(duì)大鼠急性心肌缺血的保護(hù)作用及其機(jī)制[J];中草藥;2013年08期

7 陳士林;姚輝;韓建萍;辛天怡;龐曉慧;石林春;羅q;宋經(jīng)元;侯典云;石上梅;錢忠直;;中藥材DNA條形碼分子鑒定指導(dǎo)原則[J];中國(guó)中藥雜志;2013年02期

8 李鐵柱;杜紅巖;劉慧敏;烏云塔娜;王淋;葉生晶;;杜仲果實(shí)和葉片轉(zhuǎn)錄組數(shù)據(jù)組裝及基因功能注釋[J];中南林業(yè)科技大學(xué)學(xué)報(bào);2012年11期

9 張嵐;李慶章;;新基因功能研究的整體策略[J];中國(guó)畜牧獸醫(yī);2011年05期

10 楊茵;李碩果;葉文才;江仁望;;布渣葉的化學(xué)成分研究[J];時(shí)珍國(guó)醫(yī)國(guó)藥;2010年11期

相關(guān)碩士學(xué)位論文 前1條

1 張彬;當(dāng)歸屬藥用植物及藥材的DNA條形碼鑒別研究[D];中國(guó)中醫(yī)科學(xué)院;2012年

，

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