【作者】 强玮；

【导师】 廖志华； 陈敏；

【作者基本信息】 西南大学 ， 植物学， 2012， 硕士

【摘要】 托品烷类生物碱(Tropane alkaloids, TAs)是一类主要提取自茄科植物的小分子含氮化合物,它提供了临床上常用的抗胆碱药物莨菪碱(hyoscyamine)和东莨菪碱(scopolamine),广泛用于用于镇痛、麻醉、抗晕动药、治疗帕金森症、改善微循环、戒毒脱瘾、治疗农药中毒等,市场需求十分巨大。由于工业合成成本太过昂贵,目前莨菪碱和东莨菪碱主要从茄科植物的颠茄(Atropa belladonna)、莨菪(Hyoscyamus niger)和曼陀罗(Datura stramonium)中提取,产量有限,其中颠茄是中国药典规定的唯一TAs药源植物(国家药典委员会2011版),开展代谢工程以提高颠茄中TAs含量具有十分重要的意义。本研究采用RACE方法克隆了药用植物颠茄的托品酮还原酶(?)(tropinone reductase I, TRI)基因cDNA全长并进行了详细的生物信息学分析,同时构建了该基因的植物组织表达谱和诱导表达谱；原核表达验证酶活后,构建植物表达载体遗传转化颠茄获得转基因发根,经过液体发酵培养至指数生长末期,检测总托品烷生物碱含量和相应的AbTRI基因表达量。最终结果表明：颠茄中AbTR1作为一个控制点酶,它的过量表达能够极大的促进代谢流向莨菪碱和东莨菪碱方向流动,从而显著地提高总托品烷生物碱的产量。TRI催化托品酮分子上羰基的还原生成α-托品醇(托品),是托品烷生物碱合成途径中间分支处的控制点酶。在莨菪中，TRI基因特异性的仅在根的内皮层和外皮层中强烈表达,曼陀罗和三分三(Anisodus acutangulus)的TRI基因也是克隆至培养的根组织。本研究即以颠茄根为材料,采用RACE技术,基于同源性克隆的方法获得了AbTRI基因全长cDNA,为1079bp,包含一段长度为819bp的CDS、14bp的5’UTR和234bp的3’UTR;polyA长度为13bp,终止密码子为TAA；其编码区编码一段272个氨基酸残基的蛋白序列(AbTRI),预测其分子量大小为29.3KDa,等电点为6.05。对推断的AbTRI基因编码氨基酸序列进行生物信息学分析,结果表明,AbTRI与已报道的茄科其它物种的托品酮还原酶氨基酸序列相似性均在80%以上,而且整体上都很保守,说明它们的TRIs从共同的祖先蛋白开始分化发生在相对较近的时间里；来源于茄科植物、十字花科植物、单子叶植物、细菌的TRIs在发育进化树上明显聚为4支,其中AbTRI与同科不同属的植物三分三TRI的亲缘关系最近,其次是莨菪；保守结构域预测发现AbTRI的氨基酸序列中存在典型的短链脱氢酶(short-chain dehydrogenase/reductase, SDR_c)特征性结构域,这与曼陀罗和莨若的TRIs已经被鉴定属于SDR家族这一事实相吻合；进一步的三级结构预测发现,在AbTRI的N端SDR所需要的NADPH结合区域,存在SDR优先结合NADPH的保守氨基酸残基Lys31和Arg53,同时在C端底物托品酮结合区域也发现了两个决定托品酮分子以正确的取向结合在TRI酶蛋白上的保守氨基酸残基His112和Va1168。组织表达谱和诱导表达谱分析表明,该基因强烈地在颠茄成熟叶中表达,花苞和须根也有少量表达,其它部位表达微弱,其表达水平强烈地受ABA、ASA、MeJA、UV-B的诱导和SA的抑制,溶剂乙醇也能起到强烈的诱导效果。以pET28/大肠杆菌Rosetta为诱导表达系统对AbTRI基因进行原核表达功能验证,经1mM IPTG诱导培养4h, AbTRI重组蛋白含量达到了细胞总蛋白的50%以上,主要以包含体形式存在,其分子量约为30Kda,与预测的相符。对AbTRI重组蛋白包涵体进行纯化并透析复性后,以托品酮为反应底物,在NADPH存在下,检测RcTYDC重组蛋白的催化活性,利用HPLC法检测到了反应产物托品的生成,表明本研究克隆获得了颠茄的功能AbTRI基因。构建携带AbTRI基因的植物高效表达载体p1304+-AbTRI转化“卸甲”的根癌农杆菌C58C1获得工程菌C58Cl-p1304+-AbTR],遗传转化颠茄叶片,经检测rolB、rolC、Hygr和AbTRI基因后成功筛选出7个转基因发根系,分别进行液体培养基扩大培养然后检测托品烷生物碱(TAs)含量和基因表达量。在检测的7个转基因发根系中,有4个发根系的总TAs含量得到了大幅度的提高,依次为T9>T16>T23>T11,19的总TAs含量比空菌发根对照和空载发根对照分别提高了3.2倍和8.3倍,莨菪碱的提高幅度平均要高于东莨菪碱；另外3个发根系出现了不同程度的基因沉默,TAs含量显著低于对照。利用实时荧光定量PCR技术检测这七个发根系中相应的AbTRI基因表达量,结果与生物碱含量检测的结果基本一致：四个生物碱含量提高的发根系(T9、T16、T23、T11)的基因表达水平都显著高于对照和其它转基因发根的表达水平；而三个低含量发根系(T10、T5、T15)的基因表达水平也显著低于对照,说明确实发生了基因共抑制现象。本研究的实验结果证明了在颠茄中过量表达AbTRI能够促进代谢流流向目的产物并显著提高总TAs含量,这为进一步开展颠茄的TAs代谢工程提供了新的候选基因和理论支持,结合本实验室之前的研究成果,在超量表达TAs途径上下游限速酶基因PMT和H6H的颠茄中进一步超量表达TRI,有可能最大限度的开发TAs途径的生物碱合成能力,培育出东莨菪碱超高产颠茄株系,为相应行业提供优质药源材料。更多还原

【Abstract】 Tropane alkaloids (TAs) are a class of low molecular weight nitrogen-containing substances mainly extracted from Solanaceae, which offer clinically commonly used anticholine drugs hyoscyamine and scopolamine widely for the pain relief, anesthesia, refraining drug addiction, control of motion sickness and cure of Parkinson’s disease with an enormous maket damands. Due to the expensive cost price in industral synthesis, hyoscyamine and scopolamine for now are acquired mainly by extracting from three Solanaceae plants Atropa belladonna, Hyoscyamus niger and Datura stramonium, and the production is limited, among which A. belladonna is the only commercial plant species (authorized by pharmacopoeia of The People’s Republic of China2011) that can be used to extract TAs, so it is of great significance to carry out metabolic engineering to enhance TAs contents in A. belladonna. In this study, we cloned the full-length cDNA of tropinone reductase I (TRI) from medicinal plant A. belladonna using the RACE method and made a detailed bioinformatic analysis, simultaneously expression profiles of AbTRI in different tissues and induced expression profiles were constructed; after activity validation through prokaryotic expression, the plant expression vector was constructed to genetically transform belladonna to obtain transgenic hairy root, and then liquid cultured to the end of the exponential growth to detect the total TAs content and the corresponding AbTRI gene expression. The final results showed that AbTRI functioning as a check point enzyme in A.belladonna, its overexpression can greatly promote the flow of metabolic flux towards hyoscyamine and scopolamine, thus significantly enhancing the production of the total TAs. TRI is the check-point enzyme which locates at the middle branch of TAs biosynthetic pathway, catalyzing the reduction of carbonyl in tropinone molecules to generate α-hydroxytropane (tropine). HnTRI gene in H. niger plant is strongly expressed only in the root inner cortex and outer cortex with specificity, and the TRIs in D. stramonium and Anisodus acutangulus are also cloned from the cultural root tissue. In this study, A. belladonna root was adopted for material and we successed in obtaining the full-length cDNA of AbTRI gene of1079bp based on homology cloning method, using RACE technology. The cDNA consists of a coding sequence of819bp,5’UTR of14bp,3’UTR of234bp and polyA of13bp with termination codon of TAA. The coding region encodes a272-amino-acid-residues protein (AbTRI) which was predicted to have a molecular weight of29.3KDa and an isoelectric point of6.05.Bioinformatics analysis of the deduced amino acid sequence of AbTRI showed that the AbTRI shared an amino acid sequence similarity of more than80percents with other Solanaceae species reported, and on the whole is very conservative, indicating that their TRIs start to differentiate from a common ancestor protein occurred in relatively recent time; in phylogenetic tree, TRIs from the Solanaceae, Brassicaceae, Monocots and Bacteria are clearly clustered into four branches, among which AbTRI have a closest genetic relationship with TRI from A. acutangulus which belongs to different genus from A. belladonna’s and followed by H. niger TRI. Through conserved domain prediction, we found in AbTRI amino acid sequence a typical characteristic domain of short-chain dehydrogenase (short-chain dehydrogenase/reductase, SDR_c), which is consistent with the fact that TRIs from D. stramonium and H. niger have been identified as members of the SDR family. Further tertiary structure prediction found that there is binding region of NADPH in the N-terminal SDR of AbTRI, and the conserved amino acid residues Lys31and Arg53can prioritily combined to NADPH. At the same time, it found out that there are conservative amino acid residues His112and Va1168at the C-terminal end of tropine ketone binding region, which can make sure that tropinone combined to TRI protein correctly. Tissue Expression and induction expression analysis showed that this gene specifically expressed in A. belladonna old leaves, little expressed in flowers and roots, and slightly expressed in other tissues. At the same time, its expression level was strongly affected by the ABA, ASA, MeJA, UV-B and ethanol induction and SA inhibition.Take PET28/E. coli as genealogical tree to prokaryotically identify the function of AbTRI gene. Functional verification induced by1mM IPTG induction for4h,, AbTRI recombinant protein content of more than50%of the total cellular protein, mainly in the form of inclusion bodies, the molecular weight of about30Kda consistent with the forecast. Recombinant protein inclusion bodies were purified on AbTRI and dialysis refolding, atropine ketone as a substrate in the presence of NADPH, the detection of the catalytic activity of the RcTYDC recombinant protein by HPLC detected the formation of reaction products tropine, show that the The cloned Belladonna’s the function AbTRI gene.To build carrying the A6TRI gene of a plant effective expression vector p1304+-AbTRI Agrobacterium tumefaciens C58C1was transformed into the "Resurrection" Engineering bacteria C58Cl-p1304+-AbTRI genetic transformation of belladonna leaves by detection on rol B of rolC Hygr and AbTRI, gene after successful screening seven transgenic roots, respectively, the liquid medium to expand cultivation and then detect the content of tropine the alkane alkaloids (TAs) and gene expression.5TAs content of the hair root has been greatly improved in the detection of seven genetically modified hair roots, followed by the T9> T16> of T23> T11, T9TAs content than the empty bacteria hair root control and no-load The hair root control were increased by3.2times and8.3times, the improved rate of scopolamine on average higher than that of scopolamine; three roots appear with varying degrees of gene silencing of TAs were significantly lower than the control. The AbTRI seven rounds of the roots of the real-time fluorescent quantitative PCR gene expression results with the alkaloid content of the test results are basically the same:four alkaloid content to improve the hair root (T9, T16, T23, T11) of The gene expression levels were significantly higher than control and other transgenic hair root, the expression level; three low levels of hair roots (T10, T5, T15) gene expression levels were significantly lower than control, indicating that indeed the gene co-suppression.This study the experimental results show that overexpression AbTRI can contribute to the metabolic flux to the target product and significantly increase the content of total TAs in belladonna, provides a new candidate genes and theoretical support for further belladonna TAs metabolic engineering, combined with the laboratory before the research results in the overexpression of TAs way downstream speed limit further overexpression of TRI gene the pmt and h6h of belladonna alkaloid synthesis capabilities can maximize the potential development TAs way, and encouraged scopolamine ultra-yielding the belladonna lines, to provide quality drug source material for the corresponding industries.更多还原