紫外诱导圆锥铁线莲中吲哚生物碱合成机理研究及CtIPT的克隆与原核表达分析

发布时间：2017-12-26 15:54

本文关键词：紫外诱导圆锥铁线莲中吲哚生物碱合成机理研究及CtIPT的克隆与原核表达分析　出处：《浙江大学》2017年硕士论文　论文类型：学位论文

【摘要】：中国是药用植物资源最丰富的国家之一,而药用植物在人类疾病预防和治疗中占有重要地位。UV-B对药用植物的生长发育和次生代谢影响很大,但是,不同药用植物对UV-B的具体响应情况有所不同,许多机理有待进一步发现。吲哚生物碱是药用植物中非常具有研究价值的一类次生代谢产物,在UV-B逆境下含量增加,不仅能够增强植物自我保护能力以抵御逆境胁迫,还具有细胞毒性、抗癌、抗病毒、抗疟和抗炎等功效,能够直接增加植物的药用价值。细胞分裂素是由腺苷酸异戊烯基转移酶合成的一类植物激素,在UV-B逆境下含量减少,不仅能够调控药用植物生长发育使其形态学发生改变而处于抵御逆境胁迫的状态,还可能与UVR8信号通路中的某些调控因子相关,参与调控植物次生代谢产物的合成(如吲哚生物碱、香豆素、黄酮等),能够间接增加植物的药用价值。圆锥铁线莲属于毛茛科,具有非常高的药用价值。实验室前期从圆锥铁线莲中分离出了吲哚生物碱(6-hydroxyl-1H-indol-3-yl)carboxylic acid methyl ester,本课题在该研究成果的基础上进行了更深入的研究,主要研究内容和结果如下:(1)圆锥铁线莲中吲哚生物碱响应UV-B诱导的合成机理HPLC-TOF-MS/MS分析显示圆锥铁线莲经过UV-B诱导后其吲哚生物碱(6-hydroxyl-1H-indol-3-yl)carboxylic acid methyl ester 含量增加,表明圆锥铁线莲在UV-B诱导下体内吲哚生物碱合成途径呈增强状态。基于2-DE和GC-TOF-MS技术的蛋白质组学和代谢组学研究揭示了圆锥铁线莲经UV-B诱导后吲哚生物碱的合成机理,即与氨基酸代谢相关的蛋白和化合物含量显著增加,表明氨基酸代谢被激活。通过对丝氨酸脱氨酶进行酶活检测,结果显示该酶活经过UV-B诱导后显著增强,表明氨基酸代谢过程被UV-B辐射激活能够促进下游莽草酸代谢途径的增强。基于qRT-PCR技术的转录组学研究分析了圆锥铁线莲经UV-B诱导后吲哚生物碱合成途径上关键基因的变化情况,结果显示从莽草酸到色氨酸代谢过程中的8个关键基因的表达表现出上调共性;通过HPLC-TOF-MS/MS代谢组检测圆锥铁线莲经UV-B诱导后吲哚生物碱合成途径上关键化合物的变化情况,结果显示邻氨基苯甲酸盐、吲哚和色氨酸的含量都增加;通过对色氨酸合成酶进行酶活检测,结果显示该酶活经过UV-B诱导后显著增强,上述结果均表明增强了的莽草酸代谢途径能够促进吲哚生物合成途径的增强,最终为吲哚生物碱的合成奠定了基础。(2)圆锥铁线莲中腺苷酸异戊烯基转移酶的克隆与原核表达借助转录组测序,我们获得了圆锥铁线莲叶片中腺苷酸异戊烯基转移酶的EST片段,之后利用RT-PCR和cDNA末端快速扩增技术(rapid amplification of cDNAends,RACE),得到了长度为1374 bp的碱基序列,其中包括长度为332 aa的完整ORF区。接着对该序列编码的蛋白CtIPT进行理化性质预测、序列比对和系统进化树分析,结果显示该蛋白是一个分子量为37.2 kDa的亲水性蛋白,存在腺苷酸异戊烯基转移酶特征序列区域(ATP/GTP结合序列区域),且与番茄中的S1IPT3/4和拟南芥中的AtIPT3/5/7非常相似,表明该蛋白为腺苷酸异戊烯基转移酶。然后通过qRT-PCR检测UV-B诱导对CtIPT表达的影响,结果显示具有显著性抑制作用,表明CtIPT可能具有抵御紫外逆境胁迫的功能。再分别以pET-28a(+)、pGEX-4T-1和pMAL-c2X质粒为表达载体对CtIPT进行克隆,并在大肠杆菌BL21中进行原核表达,SDS-PAGE结果显示在16℃,180rpm,0.5mM IPTG诱导表达6 h的条件下均可以成功表达出目的蛋白,但是只有在BL21-pMAL-c2X-CtIPT(含MBP标签)中实现了可溶性表达。最后通过Amylose resin亲和层析柱进行分离纯化,得到了目的蛋白腺苷酸异戊烯基转移酶。
[Abstract]:Abstract: China is one of the most abundant resources of medicinal plants, and medicinal plants play an important role in the prevention and treatment of human diseases. UV-B has great influence on the growth and secondary metabolism of medicinal plants. However, the response of different medicinal plants to UV-B is different. Many mechanisms need to be further discovered. Indole alkaloids are a class of secondary metabolites is extremely valuable in medicinal plants, increase the content of UV-B in the face of adversity, not only can enhance the ability of self - protection against plant stress, also has cytotoxicity, anticancer, antiviral, antimalarial and anti-inflammatory effect, can directly increase the medicinal value of plants. Cytokinin is a kind of plant hormone synthesis by enzyme adenylate isopentenyl transferase, reduce the content of UV-B in the face of adversity, not only can adjust the growth and development of medicinal plants in the morphological changes in the resist stress state may be associated with some regulatory factors in the UVR8 signaling pathway, is involved in the regulation of synthesis of plant secondary metabolites the (such as Huang Tong, indole alkaloids coumarins, etc.), can indirectly increase the medicinal value of plants. Clematis Clematis is a species of Ranunculus, which has very high medicinal value. From the previous sweetautumn Clematis isolated indole alkaloids (6-hydroxyl-1H-indol-3-yl) carboxylic acid methyl ester, makes a further study of this topic based on the research results, the main research contents and results are as follows: (1) sweetautumn Clematis indole alkaloids in response to the HPLC-TOF-MS/MS synthesis mechanism induced by UV-B analysis showed that the cone of Clematis after the induction of UV-B (6-hydroxyl-1H-indol-3-yl) carboxylic indole alkaloids increased acid methyl ester content showed that sweetautumn Clematis induced by UV-B in vivo indole alkaloids biosynthesis pathway was to strengthen the state. Proteomics and metabonomics based on 2-DE and GC-TOF-MS technology revealed the mechanism of indole alkaloid synthesis after Clematis induction by UV-B, that is, the content of protein and compounds related to amino acid metabolism increased significantly, indicating that amino acid metabolism was activated. Through enzyme activity detection of serine deaminase, the results showed that the enzyme activity was significantly enhanced after UV-B induction, indicating that amino acid metabolism activated by UV-B radiation can promote the enhancement of downstream shikimic acid metabolism pathway. Transcriptome qRT-PCR technology research has analyzed the changes of key genes of sweetautumn Clematis induced by UV-B Indole Alkaloids Biosynthesis Pathway Based on the results showed that the expression of 8 key genes from shikimic acid to tryptophan metabolism in the process of showing up in common; changes of key compounds through the HPLC-TOF-MS/MS metabolic detection of sweetautumn Clematis after induced by UV-B indole alkaloid biosynthesis pathway. Results showed that the anthranilic acid salt, indole and tryptophan content were increased; the tryptophan synthase enzyme activity assay, the results show that the enzyme activity induced by UV-B was significantly enhanced and the results show that the shikimate pathway enhances the can enhance the indole biosynthesis pathway, and ultimately laid the foundation for the synthesis of indole alkaloids. (2) sweetautumn Clematis in adenylate isopentenyltransferase the cloning and prokaryotic expression of the transcriptome sequencing, we obtained the EST fragment of adenylate isopentenyltransferase cone of Clematis in leaves, followed by rapid amplification of RT-PCR ends (rapid and cDNA amplification of cDNAends, RACE), the the nucleotide sequence of 1374 BP in length, including the length of ORF region of 332 aa. Then predicted and analyzed the sequence alignments and phylogenetic trees on physicochemical properties of the sequence encoding the CtIPT protein, the results showed that the protein is a molecular weight of 37.2 kDa hydrophilic protein exist adenylate isopentenyltransferase (ATP/GTP binding sequence feature sequence region region), and tomato S1IPT3/4 thaliana and AtIPT3/5/7 are very similar, indicating that the protein was adenylate isopentenyltransferase. Then, the influence of UV-B induction on CtIPT expression was detected by qRT-PCR. The results showed significant inhibition effect, indicating that CtIPT might have the function of resisting UV stress. Then to pET-28a (+), pGEX-4T-1 and pMAL-c2X plasmid expression vector of CtIPT was cloned, and the prokaryotic expression in Escherichia coli BL21, SDS-PAGE results showed that 180rpm at 16 DEG C, 0.5mM, IPTG induced expression conditions of 6 h can be successfully expressed the target protein, but only in BL21-pMAL-c2X-CtIPT (including MBP label) in soluble form. At last, the target protein adenylyl isoamyl transferase was obtained by Amylose resin affinity chromatography column.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S567.19

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