甘草酸生物合成相关糖基转移酶的研究

发布时间：2018-03-25 18:36

  本文选题：糖基转移酶　切入点：甘草　出处：《北京中医药大学》2017年博士论文

【摘要】：甘草酸是甘草的主要有效成分,具有抗炎、调节免疫和抗病毒等活性,临床上已被开发成重要的治疗乙肝药物。同样,甘草酸也是重要的甜味剂,其甜度是蔗糖的150倍。目前,甘草酸生物合成途径中大多关键酶已经被成功表征,包括关键的β-香树脂醇合成酶(bAS)和两个重要的细胞色素P450酶(CYP88D6和CYP72A154),但是催化最后两步关键反应的糖基转移酶(UGT)仍然没有被表征。本研究基于深度转录组多重数据分析和多证据链表征的方法成功获得了甘草酸生物合成相关的关键UGT。具体内容如下:(1)UGT是一个庞大的超家族酶系,为获得甘草酸生物合成相关的候选UGT,本研究采用高通量转录组测序结合多重数据分析的方法筛选目标UGT。测序结果显示,本研究获得的样品的Clean bases均达到10G以上,测序深度深;样品测序数据错误率为0.02%,Q2096%,Q3090%,测序数据的正确率高,质量优;Unigenes的总量有112307个,最短长度为201 bp,最长长度为16990 bp,平均长度为703 bp,中间长度为378 bp,N50为1164,N90为272。GO分类统计显示,有大量的unigenes参与代谢过程,具有催化活性;KOG分类统计显示,参与生理活动的unigenes较多,特别是在转录、翻译、信号转导水平;KEGG通路分析显示,有415个unigenes参与次生代谢生物合成,有295个unigenes参与萜类或聚酮类代谢。该结果暗示unigenes包含了大量与研究相关的基因,为甘草酸代谢途径的解析提供了坚实的基础。将unigenes在七大数据库(包括 Nr,Nt,Pfam,KOG/COG,Swiss-prot,KEGG,GO)中进行功能注释显示,获得的unigenes共包含潜在的434个UGT基因,进一步通过差异表达分析和共表达分析等,最终筛选得到了 8个候选UGT,分别依次命名为UGT1至UGT8,用于下一步研究。(2)为获得8个候选UGT的基因信息,本研究克隆了 8个候选糖基转移酶基因并进行了生物信息学分析。结果显示,克隆的UGT1基因的cDNA全长为1524 bp,编码507个氨基酸残基,理论分子量(MW)为56.83kDa,等电点(PI)为5.58。克隆的UGT2基因的cDNA全长为1110 bp,编码370个氨基酸残基,MW为42.53kDa,PI为5.99。克隆的UGT3基因的cDNA全长为1446 bp,编码481个氨基酸残基,MW为53.85kDa,PI为5.88。克隆的UGT4基因的cDNA全长为1443 bp,编码480个氨基酸残基,MW为53.15kDa,PI为6.23。克隆的UGT5基因的cDNA全长为1422 bp,编码473个氨基酸残基,MW为53.16kDa,PI为6.94。克隆的UGT6基因的cDNA全长为1338 bp,编码445个氨基酸残基MW为49.36kDa,PI为6.29。克隆的UGT7基因的cDNA全长为1473 bp,编码490个氨基酸残基,MW为55.24kDa,PI为5.89。克隆的UGT8基因的cDNA全长为1473 bp,编码499个氨基酸残基,MW为56.12kDa,PI为6.06。所获得的8个候选UGT的亲水性均较强,蛋白结构稳定,可能并非分泌蛋白,基本处于膜外,二级结构均主要含有αα螺旋,延伸链,β折叠,无规则卷曲。结构分析表明可能均属于UGT超家族。(3)为表征获得的8个候选UGT的活性,本研究将8个候选UGT与pET32a(+)重组构建表达载体,在BL21(DE3)菌株中诱导表达,获得的粗酶用于体外催化反应。结果显示,UGT1、UGT2、UGT4、UGT5、UGT6、UGT7、UGT8 的催化产物中均没有检测到甘草酸,但惊喜的发现是,UGT3(命名为GuUGAT)能完成连续的两步苷化反应,直接催化甘草次酸产生甘草酸。为进一步确认GuUGAT的催化活性,本研究继续将GuUGAT蛋白进行纯化后再次催化分析。结果同样证实,GuUGAT具有催化甘草次酸经过连续两步苷化反应直接产生甘草酸的活性。该结果证实,我们已成功获得了具有目标活性的UGT。(4)为进一步研究新发现的GuUGAT的生化及表达特征,本研究继续对该酶的催化参数、表达方式、系统发育等进行了研究。反应动力学参数分析结果显示,GuUGAT以甘草次酸为底物的Kcatand Km分别为2.85 s-1和36.2μM,以甘草次酸单葡萄糖醛酸为底物的Kcat and Km是0.12s-1和4.3μM。基因表达分析结果显示,GuUGAT在根茎叶中均有微量表达,但在根中经盐和干旱刺激后其表达量会显著提高,相应地,甘草酸的含量也显著增加。共表达网络分析证实GuUGAT与甘草酸生物合成的关键基因SQE、SQS、bAS和CYP88D6等存在共表达关系。GuUGAT和bAS表达量分析同样显示GuUGAT和bAS表达量的变化趋势有明显的一致性。这些证据共同一致地证明GuUGAT参与了甘草酸的生物合成。系统发育分析表明,GuUGAT可能属于UGT73家族。但是值得注意的是,GuUGAT,与其他三萜UGT不一样,GuUGAT独立成支。该结果暗示GuUGAT可能为UGT家族亚支中新的代表成员。上述证据共同证实本研究发现的GuUGAT是能催化连续两步苷化反应直接使甘草次酸转化为甘草酸的一个新颖的UGT。(5)为进一步研究GuGAT潜在的催化机制,本研究通过同源序列比对分析和分子对接的方法选择了9个位点进行点突变的研究,结果显示,Q352A,H22A,W370A,E375A和Q392A能使GuUGAT的催化活性下降约60-70%,可能是GuUGAT的关键活性位点。综上所述,本研究成功获得了具有目标活性的GuUGAT,并且惊喜地发现该酶是植物重要天然产物相关UGT中首个能催化两步葡萄糖醛酸化的UGT,同时也是三萜UGT中首个能将UDP-GlcA作为糖基供体的UGT,属于全新的发现。GuUGAT的发现为甘草酸的生物全合成及甘草的遗传育种奠定了坚实的基础。
[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.

【学位授予单位】：北京中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S567.71;Q943.2
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本文编号：1664379