發(fā)布時間：2018-05-11 03:14

  本文選題：粘毛黃芩 + 轉(zhuǎn)錄組測序��； 參考：《陜西師范大學(xué)》2016年碩士論文

【摘要】：粘毛黃芩(Scutellaria viscidula Bunge)為唇形科(Labiatae)黃芩屬(Scutellaria)多年生草本植物,以干燥根入藥,主要活性成分為黃酮類化合物(如黃芩苷、黃芩素等),具有抗心腦血管疾病、抗癌、抗炎等多種生物活性。目前,藥用植物基因組數(shù)據(jù)相對匱乏,遺傳背景及其基因信息嚴(yán)重不足,導(dǎo)致大多數(shù)藥用植物活性成分合成的途徑及分子機制不清楚。隨著轉(zhuǎn)錄組測序技術(shù)的快速發(fā)展和完善,利用轉(zhuǎn)錄組測序獲得的大量基因信息數(shù)據(jù)揭示藥用植物次生代謝生物合成途徑及其關(guān)鍵酶基因克隆、表達(dá)調(diào)控等分子機制成為藥用植物研究熱點。目前,國內(nèi)對粘毛黃芩的研究主要集中在資源評價利用和部分植物表達(dá)載體的構(gòu)建等,尚不清楚藥用活性成分黃酮類化合物(如黃芩苷、黃芩素等)合成的遺傳基礎(chǔ),更缺少合成途徑中關(guān)鍵酶基因結(jié)構(gòu)及表達(dá)調(diào)控等相關(guān)信息。本研究以粘毛黃芩為材料,通過轉(zhuǎn)錄組測序構(gòu)建轉(zhuǎn)錄組數(shù)據(jù)庫,在此基礎(chǔ)上挖掘黃酮合成途徑關(guān)鍵基因,并通過熒光定量PCR分析了結(jié)構(gòu)基因和調(diào)控轉(zhuǎn)錄因子的表達(dá),結(jié)合總黃酮積累動態(tài)變化和基因表達(dá),初步探討粘毛黃芩藥用活性成分類黃酮的合成途徑及其分子機制。本研究主要獲得以下研究結(jié)果：1.以粘毛黃芩為材料,提取RNA,基于Illumina HiSeq/MiSeq 進行轉(zhuǎn)錄組測序,共獲得reads數(shù)42310834條,組裝出40052個Unigenes, Unigenes平均讀長882bp,N50讀長1577bp。將粘毛黃芩測序獲得的Unigenes同6個蛋白數(shù)據(jù)庫(NR、KO、SwissProt、Pfam、GO、KOG)和Nt核酸數(shù)據(jù)庫進行Blast比對,共有24892條(62.14%)序列得到注釋。通過與KOG數(shù)據(jù)庫的比對,粘毛黃芩基因被分到26個不同的功能組中,通過Blast比對和ESTScan軟件預(yù)測,共獲得33367條CDS記錄。在此基礎(chǔ)上建立粘毛黃芩轉(zhuǎn)錄組數(shù)據(jù)庫,為粘毛黃芩基因工程育種和活性成分形成分子機制探索提出新的研究思路和方法。2.通過對數(shù)據(jù)庫中SSR進行統(tǒng)計分析,粘毛黃芩共檢測出8925個SSRs位點,其中二核苷酸是主要的重復(fù)類型(占總SSRs的51.69%),其次是單、三核苷酸重復(fù)(29.03%,18.32%)。SSRs位點中優(yōu)勢二核苷酸基元類型是AG/CT(占總SSRs的40.78%)。SSR信息分析將為粘毛黃芩功能基因的挖掘和分子標(biāo)記輔助選擇育種等奠定了基礎(chǔ)。3.對粘毛黃芩黃酮類化合物合成相關(guān)基因代謝通路進行生物信息學(xué)分析,結(jié)果有136條注釋到苯丙烷代謝途徑,41條注釋到黃酮類合成途徑。其中注釋獲得有4條查爾酮合酶(CHS)、1條查爾酮異構(gòu)酶(CHI)、2條黃烷酮3-羥化酶(F3H)、11條苯丙氨酸解氨酶(PAL)、5條4-香豆酰輔酶A連接酶(4CL)基因。另外,在粘毛黃芩轉(zhuǎn)錄組數(shù)據(jù)庫中還發(fā)現(xiàn)與黃酮類化合物合成相關(guān)MYB轉(zhuǎn)錄因子序列(MYB)88條,堿性螺旋-環(huán)-螺旋轉(zhuǎn)錄因子序列(bHLH)34條和WD40重復(fù)蛋白轉(zhuǎn)錄因子序列(WD40)10條。4.通過NCBI數(shù)據(jù)庫和生物信息學(xué)分析,篩選和鑒定出粘毛黃芩黃酮類化合物生物合成相關(guān)基因(CHI、CHS和F3H)和轉(zhuǎn)錄因子(bHLH, MYB2)的部分序列,并以此為模板設(shè)計引物。應(yīng)用熒光定量PCR技術(shù)對上述粘毛黃芩5個黃酮類化合物生物合成相關(guān)基因在三個時期的根、莖和葉的表達(dá)量進行檢測,結(jié)果表明,三個時期根、莖和葉均檢測到基因不同程度表達(dá),CHS、F3H和CHI在7月份各部位表達(dá)量較高。另外,MYB2基因在三個時期的表達(dá)量呈現(xiàn)下降趨勢,表現(xiàn)出同一部位中的表達(dá)可能影響CHS和CHI的表達(dá),暗示對CHS的正向調(diào)控,而對CHI的負(fù)向調(diào)控,但不具顯著相關(guān)性。5.提取粘毛黃芩三個時期不同部位的總黃酮,以黃芩苷為標(biāo)準(zhǔn)品,利用紫外分光光度法測定根、莖和葉在三個不同時期總黃酮含量,結(jié)果發(fā)現(xiàn)7月份三個部位總黃酮含量顯著高于5月和9月,且均呈現(xiàn)出根莖葉。結(jié)合各時期CHS、F3H和CHI的表達(dá)量,初步顯示在7月份總黃酮含量較高時,CHS、F3H和CHI基因的表達(dá)量也較高,暗示基因表達(dá)與總黃酮含量間可能存在正相關(guān)。上述研究獲得的粘毛黃芩轉(zhuǎn)錄組數(shù)據(jù)、黃酮合成途徑關(guān)鍵基因和轉(zhuǎn)錄因子,初步探討了粘毛黃芩黃酮類化合物合成途徑關(guān)鍵基因表達(dá)與總黃酮量的關(guān)系,為進一步揭示粘毛黃芩活性成分形成分子機制和通過基因工程手段提高藥用成分含量提供依據(jù),具有重要的理論和實際意義。
[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.

【學(xué)位授予單位】：陜西師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：Q943.2;S567.239

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