本文選題：糖基轉(zhuǎn)移酶　切入點(diǎn)：甘草　出處：《北京中醫(yī)藥大學(xué)》2017年博士論文

【摘要】：甘草酸是甘草的主要有效成分,具有抗炎、調(diào)節(jié)免疫和抗病毒等活性,臨床上已被開(kāi)發(fā)成重要的治療乙肝藥物。同樣,甘草酸也是重要的甜味劑,其甜度是蔗糖的150倍。目前,甘草酸生物合成途徑中大多關(guān)鍵酶已經(jīng)被成功表征,包括關(guān)鍵的β-香樹(shù)脂醇合成酶(bAS)和兩個(gè)重要的細(xì)胞色素P450酶(CYP88D6和CYP72A154),但是催化最后兩步關(guān)鍵反應(yīng)的糖基轉(zhuǎn)移酶(UGT)仍然沒(méi)有被表征。本研究基于深度轉(zhuǎn)錄組多重?cái)?shù)據(jù)分析和多證據(jù)鏈表征的方法成功獲得了甘草酸生物合成相關(guān)的關(guān)鍵UGT。具體內(nèi)容如下:(1)UGT是一個(gè)龐大的超家族酶系,為獲得甘草酸生物合成相關(guān)的候選UGT,本研究采用高通量轉(zhuǎn)錄組測(cè)序結(jié)合多重?cái)?shù)據(jù)分析的方法篩選目標(biāo)UGT。測(cè)序結(jié)果顯示,本研究獲得的樣品的Clean bases均達(dá)到10G以上,測(cè)序深度深;樣品測(cè)序數(shù)據(jù)錯(cuò)誤率為0.02%,Q2096%,Q3090%,測(cè)序數(shù)據(jù)的正確率高,質(zhì)量?jī)?yōu);Unigenes的總量有112307個(gè),最短長(zhǎng)度為201 bp,最長(zhǎng)長(zhǎng)度為16990 bp,平均長(zhǎng)度為703 bp,中間長(zhǎng)度為378 bp,N50為1164,N90為272。GO分類統(tǒng)計(jì)顯示,有大量的unigenes參與代謝過(guò)程,具有催化活性;KOG分類統(tǒng)計(jì)顯示,參與生理活動(dòng)的unigenes較多,特別是在轉(zhuǎn)錄、翻譯、信號(hào)轉(zhuǎn)導(dǎo)水平;KEGG通路分析顯示,有415個(gè)unigenes參與次生代謝生物合成,有295個(gè)unigenes參與萜類或聚酮類代謝。該結(jié)果暗示unigenes包含了大量與研究相關(guān)的基因,為甘草酸代謝途徑的解析提供了堅(jiān)實(shí)的基礎(chǔ)。將unigenes在七大數(shù)據(jù)庫(kù)(包括 Nr,Nt,Pfam,KOG/COG,Swiss-prot,KEGG,GO)中進(jìn)行功能注釋顯示,獲得的unigenes共包含潛在的434個(gè)UGT基因,進(jìn)一步通過(guò)差異表達(dá)分析和共表達(dá)分析等,最終篩選得到了 8個(gè)候選UGT,分別依次命名為UGT1至UGT8,用于下一步研究。(2)為獲得8個(gè)候選UGT的基因信息,本研究克隆了 8個(gè)候選糖基轉(zhuǎn)移酶基因并進(jìn)行了生物信息學(xué)分析。結(jié)果顯示,克隆的UGT1基因的cDNA全長(zhǎng)為1524 bp,編碼507個(gè)氨基酸殘基,理論分子量(MW)為56.83kDa,等電點(diǎn)(PI)為5.58。克隆的UGT2基因的cDNA全長(zhǎng)為1110 bp,編碼370個(gè)氨基酸殘基,MW為42.53kDa,PI為5.99。克隆的UGT3基因的cDNA全長(zhǎng)為1446 bp,編碼481個(gè)氨基酸殘基,MW為53.85kDa,PI為5.88。克隆的UGT4基因的cDNA全長(zhǎng)為1443 bp,編碼480個(gè)氨基酸殘基,MW為53.15kDa,PI為6.23。克隆的UGT5基因的cDNA全長(zhǎng)為1422 bp,編碼473個(gè)氨基酸殘基,MW為53.16kDa,PI為6.94�？寺〉腢GT6基因的cDNA全長(zhǎng)為1338 bp,編碼445個(gè)氨基酸殘基MW為49.36kDa,PI為6.29。克隆的UGT7基因的cDNA全長(zhǎng)為1473 bp,編碼490個(gè)氨基酸殘基,MW為55.24kDa,PI為5.89。克隆的UGT8基因的cDNA全長(zhǎng)為1473 bp,編碼499個(gè)氨基酸殘基,MW為56.12kDa,PI為6.06。所獲得的8個(gè)候選UGT的親水性均較強(qiáng),蛋白結(jié)構(gòu)穩(wěn)定,可能并非分泌蛋白,基本處于膜外,二級(jí)結(jié)構(gòu)均主要含有αα螺旋,延伸鏈,β折疊,無(wú)規(guī)則卷曲。結(jié)構(gòu)分析表明可能均屬于UGT超家族。(3)為表征獲得的8個(gè)候選UGT的活性,本研究將8個(gè)候選UGT與pET32a(+)重組構(gòu)建表達(dá)載體,在BL21(DE3)菌株中誘導(dǎo)表達(dá),獲得的粗酶用于體外催化反應(yīng)。結(jié)果顯示,UGT1、UGT2、UGT4、UGT5、UGT6、UGT7、UGT8 的催化產(chǎn)物中均沒(méi)有檢測(cè)到甘草酸,但驚喜的發(fā)現(xiàn)是,UGT3(命名為GuUGAT)能完成連續(xù)的兩步苷化反應(yīng),直接催化甘草次酸產(chǎn)生甘草酸。為進(jìn)一步確認(rèn)GuUGAT的催化活性,本研究繼續(xù)將GuUGAT蛋白進(jìn)行純化后再次催化分析。結(jié)果同樣證實(shí),GuUGAT具有催化甘草次酸經(jīng)過(guò)連續(xù)兩步苷化反應(yīng)直接產(chǎn)生甘草酸的活性。該結(jié)果證實(shí),我們已成功獲得了具有目標(biāo)活性的UGT。(4)為進(jìn)一步研究新發(fā)現(xiàn)的GuUGAT的生化及表達(dá)特征,本研究繼續(xù)對(duì)該酶的催化參數(shù)、表達(dá)方式、系統(tǒng)發(fā)育等進(jìn)行了研究。反應(yīng)動(dòng)力學(xué)參數(shù)分析結(jié)果顯示,GuUGAT以甘草次酸為底物的Kcatand Km分別為2.85 s-1和36.2μM,以甘草次酸單葡萄糖醛酸為底物的Kcat and Km是0.12s-1和4.3μM�；虮磉_(dá)分析結(jié)果顯示,GuUGAT在根莖葉中均有微量表達(dá),但在根中經(jīng)鹽和干旱刺激后其表達(dá)量會(huì)顯著提高,相應(yīng)地,甘草酸的含量也顯著增加。共表達(dá)網(wǎng)絡(luò)分析證實(shí)GuUGAT與甘草酸生物合成的關(guān)鍵基因SQE、SQS、bAS和CYP88D6等存在共表達(dá)關(guān)系。GuUGAT和bAS表達(dá)量分析同樣顯示GuUGAT和bAS表達(dá)量的變化趨勢(shì)有明顯的一致性。這些證據(jù)共同一致地證明GuUGAT參與了甘草酸的生物合成。系統(tǒng)發(fā)育分析表明,GuUGAT可能屬于UGT73家族。但是值得注意的是,GuUGAT,與其他三萜UGT不一樣,GuUGAT獨(dú)立成支。該結(jié)果暗示GuUGAT可能為UGT家族亞支中新的代表成員。上述證據(jù)共同證實(shí)本研究發(fā)現(xiàn)的GuUGAT是能催化連續(xù)兩步苷化反應(yīng)直接使甘草次酸轉(zhuǎn)化為甘草酸的一個(gè)新穎的UGT。(5)為進(jìn)一步研究GuGAT潛在的催化機(jī)制,本研究通過(guò)同源序列比對(duì)分析和分子對(duì)接的方法選擇了9個(gè)位點(diǎn)進(jìn)行點(diǎn)突變的研究,結(jié)果顯示,Q352A,H22A,W370A,E375A和Q392A能使GuUGAT的催化活性下降約60-70%,可能是GuUGAT的關(guān)鍵活性位點(diǎn)。綜上所述,本研究成功獲得了具有目標(biāo)活性的GuUGAT,并且驚喜地發(fā)現(xiàn)該酶是植物重要天然產(chǎn)物相關(guān)UGT中首個(gè)能催化兩步葡萄糖醛酸化的UGT,同時(shí)也是三萜UGT中首個(gè)能將UDP-GlcA作為糖基供體的UGT,屬于全新的發(fā)現(xiàn)。GuUGAT的發(fā)現(xiàn)為甘草酸的生物全合成及甘草的遺傳育種奠定了堅(jiān)實(shí)的基礎(chǔ)。
[Abstract]:Glycyrrhizic acid is the main active ingredient of licorice, has anti-inflammatory, immunomodulatory and antiviral activity, clinical treatment of hepatitis B drugs have been developed into an important. Similarly, glycyrrhizic acid is an important sweetener, which is 150 times sweeter than sugar. At present, most of glycyrrhizic acid biosynthesis key enzyme has been successfully characterized. Including the key of beta amyrin synthase (bAS) and the two important cytochrome P450 enzymes (CYP88D6 and CYP72A154), but the last two steps of key catalytic glycosyl transferase reaction (UGT) has not been characterized. The research method of multiple deep transcriptome data analysis and evidence chain based on representation of success the key UGT. of glycyrrhizic acid biosynthesis related to the specific contents are as follows: (1) UGT is a large superfamily of enzymes, as a candidate UGT for glycyrrhizic acid biosynthesis, high-throughput transcriptome sequencing results by this study The method of data analysis of multiple target screening UGT. sequencing results showed that the samples obtained in this study were Clean bases above 10G, sequencing depth; sample sequencing data error rate was 0.02%, Q2096%, Q3090%, the correct rate of sequencing data of high quality; the amount of Unigenes is 112307, the minimum length is 201 BP, the longest length is 16990 BP, the average length of 703 BP, the middle length is 378 BP, N50 1164, N90 272.GO classification statistics show that there are a lot of unigenes involved in metabolic process, catalytic activity; KOG classification statistics, ginseng and physiological activities of unigenes more, especially in the transcription, translation. The expression of signal transduction pathway; KEGG analysis showed that 415 unigenes is involved in the biosynthesis of secondary metabolism, there are 295 unigenes involved in terpenoid or polyketide metabolism. The results suggest that unigenes contains a large number of related research for glycyrrhizic acid metabolism genes. Provide a solid foundation for way analysis. Unigenes in seven database (including Nr, Nt, Pfam, KOG/COG, Swiss-prot, KEGG, GO) in functional annotation showed that the unigenes contains 434 potential UGT genes, further through the analysis of differential expression and co expression analysis, finally screened 8 candidate UGT, respectively named UGT1 and UGT8, for the next step of the research. (2) to obtain 8 candidate gene information of UGT, this study cloned 8 candidate glycosyltransferase gene and bioinformatics analysis. The results showed that the full-length cDNA cloned UGT1 gene was 1524 BP, encoding 507 amino acid residues, theoretical molecular weight (MW) was 56.83kDa, isoelectric point (PI) for the full-length UGT2 5.58. clone of cDNA is 1110 BP, encoding 370 amino acid residues, MW 42.53kDa, PI UGT3 based 5.99. full-length cDNA clone for the 1446 BP encoding 481 Amino acid residues, MW 53.85kDa, PI cDNA UGT4 full-length 5.88. clone was 1443 BP, encoding 480 amino acid residues, MW 53.15kDa, PI cDNA UGT5 full-length 6.23. clone was 1422 BP, encoding 473 amino acid residues, MW 53.16kDa, PI UGT6 full length cDNA 6.94. clone was 1338 BP, encoding 445 amino acid residues of MW 49.36kDa, PI UGT7 cDNA full-length 6.29. clone was 1473 BP, encoding 490 amino acid residues, MW 55.24kDa, PI cDNA UGT8 full-length 5.89. clone was 1473 BP, encoding 499 amino acid MW 56.12kDa, residues PI as hydrophilic 8 candidate UGT 6.06. obtained were strong, stable protein structure, may not be a secreted protein, basically in the film, two structures are mainly containing alpha alpha helix, extended chain, beta folding, random coil structure. The analysis shows that may belong to UGT super family (3). 8 candidate UGT for the characterization of the activity, this study will be 8 candidate UGT and pET32a (+) to construct a recombinant expression vector in BL21 (DE3) expression strain, crude enzyme obtained for in vitro catalytic reaction. The results showed that UGT1, UGT2, UGT4, UGT5, UGT6, UGT7, no the catalytic product of UGT8 detected glycyrrhizic acid, but the surprise is that UGT3 (named GuUGAT) to complete the two step Ganhua continuous reaction, direct catalytic glycyrrhetinic acid produced glycyrrhizic acid. To further confirm the catalytic activity of GuUGAT, this study will continue to analysis GuUGAT protein were purified by again. The same results GuUGAT has confirmed that the catalytic glycyrrhetinic acid after two step reaction Ganhua directly produces activity of glycyrrhizic acid. The results showed that we have successfully obtained the target activity of UGT. (4) for further study of expression and characteristics of the new GuUGAT, this research continues Expression of catalytic parameters of the enzyme, phylogenetic analysis results were studied. The kinetic parameters showed that GuUGAT with glycyrrhetinic acid as the substrate of Kcatand Km were 2.85 S-1 and 36.2 M, with glycyrrhetinic acid mono glucuronide as substrate, Kcat and Km is the result of the analysis showed that the expression of 0.12s-1 and 4.3 M. gene, GuUGAT in both roots and leaves trace expression in roots, but the salt and drought after the stimulation of the expression will be significantly improved, accordingly, the content of glycyrrhizic acid was also increased significantly. Co expression network analysis confirmed the key genes SQE, GuUGAT and glycyrrhizic acid biosynthesis of SQS, bAS and CYP88D6 co the expression of.GuUGAT and bAS between GuUGAT and bAS content analysis also shows the trend of expression was remarkably consistent. These common evidence to prove that the GuUGAT involved in the biosynthesis of glycyrrhizic acid. The phylogenetic analysis indicated that G UUGAT may belong to UGT73 family. But it is worth noting that GuUGAT, unlike the other three from UGT, GuUGAT independent branch. The results suggest that GuUGAT may be as the representative of new members in the UGT family. The sub branch of evidence confirmed that the study found that the GuUGAT is capable of continuous catalytic reaction directly to step two in licorice acid into a novel UGT. glycyrrhizic acid (5) to study the catalytic mechanism of GuGAT potential further, this research method through homologous sequence analysis and molecular docking chose 9 loci of point mutation results showed that Q352A, H22A, W370A, E375A and Q392A could increase the catalytic activity of GuUGAT the loss of about 60-70%, may be the key to the active site of GuUGAT. In conclusion, this study successfully obtained with active GuUGAT, and surprised to find that the enzyme can catalyze the first two plant natural products UGT The step glucuronated UGT is also the first UGT in the three terpenoid UGT, which can take UDP-GlcA as a sugar donor. It belongs to the new discovery of.GuUGAT. It has laid a solid foundation for the biological synthesis of glycyrrhizic acid and the genetic breeding of Glycyrrhiza uralensis.

【學(xué)位授予單位】：北京中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S567.71;Q943.2
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本文編號(hào)：1664379