【作者】 李凤岚；

【导师】 马小军； 邱德有；

【作者基本信息】 北京协和医学院 ， 生药学， 2009， 博士

【摘要】 紫杉醇(taxol)是Wall等首次从短叶红豆杉(Taxus Brevifolia)树皮中分离得到的一种重要的次生代谢产物,是迄今为止最具抗癌活性的天然化合物之一。本论文围绕紫杉醇的生物合成过程展开了以下研究：1、紫杉烷13α-,14β-羟基化酶基因调控研究紫杉醇生物合成全过程约20步酶促反应。其中,紫杉烷13a-羟基化酶(简称130H)是紫杉醇生物合成途径的关键羟化酶。而紫杉烷14p-羟基化酶(简称140H)存在于包括紫杉醇在内的各种C-13氧取代的紫杉烷生物合成分支途径中。如果能够抑制紫杉醇生物合成的分支途径,则有可能提高该物质的生物合成产量。应用本试验室构建好的紫杉烷13α-羟基化酶基因正向表达载体、14-p羟基化酶基因反义RNA表达载体、14-p羟基化酶基因RNAi表达载体和几种可能对紫杉醇生物合成产生影响的miRNA表达载体,以曼地亚红豆杉(Taxusxmedia)愈伤组织细胞为材料进行了转化实验,并运用southern blot. RT-PCR、HPLC等技术进行了分析,结果如下：(1)建立了一套将转基因方法应用于红豆杉培养细胞的高效、完整的转化体系,对红豆杉愈伤组织细胞的转化成功率达到了100%。并探讨了几种影响转化效率的因素。(2)利用14-β羟基化酶基因RNAi表达载体(pZ14aRNAi、pZ14bRNAi)以及14-p羟基化酶基因反义RNA表达载体(pZl4c)进行了转化红豆杉细胞实验。Southern检测初步证实外源基因已经整合到了植物基因组中；RT-PCR分析结果表明,内参基因β-actin在转基因和非转基因细胞中表达水平基本一致,而140H基因在转基因细胞系中的表达水平相对非转基因细胞系显著下降。HPLC结果分析显示,转基因的红豆杉细胞与对照相比三种C-14氧取代紫杉烷(yunnanxane, sinenxan A, sinenxan C)的含量发生了较大的改变。其中主要的紫杉烷Sinenxan A含量明显下降,三种紫杉烷在转基因红豆杉细胞中的总含量呈显著下降趋势。(3)利用pC130H载体转化红豆杉细胞实验。GUS检测,荧光观察及southern blot分析初步证明pC130H载体(13α-羟基化酶基因正向表达载体)成功转化红豆杉细胞。RT-PCR结果表明,内参基因β-act in在转基因和非转基因细胞中表达水平一致,而13OH基因在转基因细胞系中的表达水平显著,却在对照中未表达。HPLC检测三种C-14氧取代紫杉烷类化合物的总量在转基因细胞中均略微有所下降。(4)其他转化红豆杉细胞的实验。本研究还进行了pC13OH、pZ14a共转化,pC13OH、pZ14b共转化,pC13OH、pZ14c共转化,pC130H、pCO169d共转化,pC13OH、pE415共转化红豆杉细胞的实验.另外，利用构建的几种miRNA表达载体如169d、pROK2-JERFs也对曼地亚红豆杉细胞进行了转化。对转化后的样品进行分析显示,共转化试验及几种niRNA表达载体均未对紫杉醇生物合成途径产生较大影响。2、茉莉酸甲酯诱导下红豆杉细胞中蛋白质的表达已有的研究表明采用茉莉酸甲酯(MJ)诱导可以有效的提高紫杉醇及紫杉烷类物质在红豆杉细胞中的含量。研究该物质诱导的作用机制,可以更全面了解茉莉酸甲酯对红豆杉细胞基因表达的影响,找到可能与紫杉醇生物合成相关的重要基因或转录因子。本试验利用茉莉酸甲酯诱导中国红豆杉(Taxus chinensis)愈伤组织细胞,采用双向电泳技术、质谱技术对茉莉酸甲酯诱导15天后细胞中的蛋白质变化进行了分析,找到22个差异表达的蛋白。经过肽质量指纹分析、数据库查询,初步断定CK(对照组)中的219号、231号、156号、127号蛋白分别与actin2(一种细胞骨架蛋白)、enolase(烯醇酶,一种参与糖酵解系统的酶,催化2-磷酸甘油酸失水而生成磷酸烯醇丙酮酸反应的酶)、F1-ATP酶α亚单位、maturase K(一种成熟酶)具有同源性。经过茉莉酸甲酯诱导后,该四种蛋白的表达量均有不同程度的降低。这可能与茉莉酸甲酯诱导引起红豆杉细胞的凋亡有关。另外,有10个蛋白与数据库中所有蛋白的同源性都很低,可能代表了中国红豆杉中与茉莉酸甲酯诱导相关的未知蛋白。3、抗凋亡抑制剂DEVD-Capaselll对紫杉醇生物合成的影响DEVD-Capaselll是一种抗细胞凋亡抑制剂,在红豆杉细胞培养基加入该物质则有可能抑制细胞的凋亡从而达到提高紫杉醇生物合成产量的目的。以曼地亚红豆杉愈伤组织作为实验材料,采用添加200μMMj(茉莉酸甲酯)的6,7-V作为培养基,试验组加入501μM的Ac-DEVD-CMK,分别在继代9天和21天后取样,观察细胞的颜色、生长情况等,并运用HPLC方法对细胞中的紫杉烷成分的种类及含量进行分析。结果发现：MJ诱导后并未在曼地亚红豆杉细胞中检测到紫杉醇的变化,说明本试验中细胞的凋亡并不是主要由于紫杉醇的积累而是由于MJ引发的反应；在细胞生长周期的早期阶段加入Ac-DEVD-CMK,不能阻止Caspase的活性,未能抑制愈伤组织细胞的凋亡。4、未知化合物的鉴定无论是在转基因还是MJ诱导试验过程中,我们均检测到了一种未知化合物,并对该化合物进行了纯化试验。该物质在HPLC色谱图上的保留时间比紫杉醇的保留时间延迟了大约0.4min。将该物质的二级质谱图与分子量类似的已知化合物的谱图进行比对,没有发现完全吻合的质谱特征。经过HPLC-HRMS分析得到该未知化合物分子量为851.39747,在Xcalibur version2.2软件中计算得到最可能是C46H55O10N6等化合物中的一种。结论：(1)本实验建立了一套高效转基因体系,并应用转基因技术成功地抑制了曼地亚红豆杉细胞系中14OH基因的表达,C-14位氧化紫杉烷类含量在转基因细胞系中均有所下降；(2)在采用茉莉酸甲酯(MJ)诱导中国红豆杉愈伤组织细胞的试验中,鉴定了四种并发现了10个可能与茉莉酸甲酯诱导有关的蛋白；(3)抗凋亡抑制剂DEVD-CapaseⅢ未对紫杉醇生物合成产生较大的影响；(4)在实验过程中检测到了一种有可能是未报道的化合物,并进行了初步的纯化和分析。更多还原

【Abstract】 SummaryTaxol (generic name paclitaxel), a complex diterpenoid derived by Wall from the Pac-ific yew (Taxus brevifolia) tree, has been approved as an important antitumor and antile-ukemic drug. Several experiments around the biosyntheses of Taxol were performed.1. Studies on Regulation of the Gene Expression of Taxol BiosynthesisIt is estimated that the biosyntheses of Taxol in vivo, from the universal diterpenoid precursor geranylgeranyl diphosphate, involves at least20distinct enzymatic steps with a similar number of taxoid intermediates. Many metabolites are intermediates of parallel or divergent taxoid pathways. The cytochrome P450taxoid14β-hydroxylase (14OH) is als-o thought to operate early in the pathway but to direct the formation of sideroute C-14o-xygenated taxoids. The cytochrome P450taxoid13a-hydroxylase (13OH) is considered to catalyze an early Taxol pathway oxygenation step that leads to C-13oxygenated taxoi-ds such as Taxol, baccatinⅢ and10-deacetylbaccatinIII. If side-route C-14oxygenated taxoids is silenced, the biosynthesis production of taxol may increase.To determine the e-ffect of suppression of this side-route on taxoids production, an antisense and RNAi appr-oach were used in Taxus×media cell line. The vector of13OH gene and other microRN-A vectors which could affect the biosynthesis pathway of paclitaxel were introduced into the cell line too. Southern blot, RT-PCR and HPLC were used to analyze the transgenic cells.We got the results as follow:(1) A high efficient transformation system was constructed in our study. The system provided a feasible way to promote the yield of taxol in biosynthesis pathway and the transformation rate can reach100%. Several important factors affecting the efficiency of transformation were summarized.(2) The dsRNA and antisense RNA expression constructs were introduced into cells by Agrobacterium tumefaciens-mediated transformation. Southern blot analysis of transgenic Taxus×media cells revealed that the transgenic cells possessed the dsRNA and anti-RNA unit. The expression of housekeeping gene β-actin was similar in the transgenic cell lines and the controls but the levels of14OH gene transcripts were significantly reduced in the transgenic cell lines by the silencing strategies, as determined by semiquantitative RT-PCR. Analysis of5major taxoids by HPLC revealed that the total yield of three major C-14oxygenated taxoids (yunnanxane, sinenxan A, sinenxan C) was reduced dramatically in these silenced cell lines as compared to non-transgenic controls.(3) The pC13OH vector which harbored the13OH gene was constructed and introduced into the Taxus cells. GUS activity determination, GFP expression and southern blot analysis of transgenic cells revealed that the exogenous gene was integrated into the Taxus genome. RT-PCR analysis showed that the levels of β-actin gene transcripts were the same in the transgenic cell lines as that in the controls, but the expressions of13OH gene were notably increased in the transgenic cell lines. Analysis of HPLC revealed that the total yield of three C-14oxygenated taxoids was reduced slightly in transgenic cell lines as compared to the controls.(4) Several co-transformation experiments (such as pC13OH and pZ14a, pC13OH andpZ14b, pC13OH and pZ14c, etc) and other transgenic experiments using microRNA vectors were performed in the study. Analysis indicated that the co-transformation and those microRNA vectors didn’t affect the biosynthesis pathway of taxol remarkably.2. Analysis of Protein profile in Taxus sp. Cells Treated with Methyl JasmonateThe previous research reported that MJ (Methyl jasmonate) is a plant growth regulator that plays an important role in regulating the biosynthesis of plant secondary metabolites and can promote the production of taxol efficiently, as well as other taxoids. To study the operation mechanism of MJ, we can know about the MJ’s effect on the genes involved in the biosynthesis of taxol roundly and may find some new key genes and transcription factors. The callus of Taxus chinensis treated with MJ was used as material. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was used to obtain the protein profiles of the taxus cells induced by MJ on day15. Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) technique was employed to generate the peptide mass fingerprinting (IMF) of the differentially expressed proteins.22differentially expressed proteins were found and database search was performed. Most of proteins hit putative proteins, and only the proteins marked with219,231,156and127in CK’s gel (the control) showed some similarity with actin2, enolase, Fl-ATPase alpha subunit and maturase K proteins respectively in the database. The levels of these proteins decreased in the cells induced by MJ. Ten proteins have low similarity with those in the database and may be the unknown ones related to the induction of MJ.3. The effect of DEVD-Capaselll on the biosynthesis of paclitaxel The apoptosis can be suppressed by Capase-3inhibitor. If it is applied in the culturing of taxus, the yield of taxol may increase. To investigate the effect of DEVD-CapaseⅢ (Capase-3inhibitor) on the biosynthesis of paclitaxel, the Taxus media cells were treated with50μM Ac-DEVD-CMK. The6,7-V medium supplented with200μM MJ was used in both the test group and the controls. The materials were harvested on day9and21after subculturing. The changes of cells (such as colour and growth rate) were observed and HPLC was performed too. Results showed that taxol was not detected in the Taxus cells treated with MJ. We supposed that is not paclitaxel to induce apoptosis but MJ did and applying the Ac-DEVD-CMK in the early stage of the cell culture didn’t block the caspase pathway.4. Identification of the unknown compoundIn our study, an unknown compound was found by HPLC and we try to purify it. The compound’s retention time delays0.4min compared to the taxol’s on the HPLC chromatogram.The results of MS/MS indicated that it may be an unreported compound. HPLC-HRMS analysis revealed that its molecular weight is851.39747and it may be C46H55O10N6or another compound among those calculated by Xcalibur version2.2.Conclusion:(1) A high efficient transformation system was constructed in our study. The levels of14OH gene was successfully suppressed by the silencing transgenic strategies and HPLC revealed that the total yield of three major C-14oxygenated taxoids was reduced dramatically in these silenced cell lines as compared to non-transgenic controls.(2) Four proteins were discriminated and ten unknown ones induced by MJ were found.(3) Applying the Ac-DEVD-CMK in the early stage of the cell culture didn’t block the apoptosis of taxus cells.(4) An unknown compound which may be unreported was found in all of our experiments and we purified and analyzed it primordially.更多还原