Engineering Terpenoid Indole Alkaloids Biosynthetic Pathway in Catharanthus roseus Hairy Root Cultures by Overexpressing the Geraniol 10-hydroxylase Gene

Catharanthus roseus contains important anti-tumor terpenoid indole alkaloids (TIAs) such as vinblastine and vincristine. Cytochrome P450 enzyme geraniol 10-hydroxylase (G10H) is a putative rate-limiting enzyme involved in the TIAs biosynthetic pathway in C. roseus. In this study the g10h gene driven by the cauliflower mosaic virus 35S (CaMV 35S) promoter was introduced into C. roseus through Agrobacterium-mediated transformation. The integration and overexpression of the target gene (g10h) in hairy root lines were confirmed by polymerase chain reaction and RT-QPCR analysis respectively. Overexpression of g10h in transgenic hairy root lines significantly enhanced the accumulations of monomeric alkaloid ajmalicine and dimeric alkaloids, vincristine and vinblastine. Total TIAs production in hairy roots reached (9.51) mg/g DW, over 3-fold higher than that in the untransformed root lines. This is the first report that engineering of g10h into TIAs-producing plant species results in significant enhancement of TIAs accumulation in cultured hairy roots. This study demonstrates that the putative rate-limiting step catalyzed by G10H is indeed the real rate-limiting step involved in the TIAs biosynthetic pathway in C. roseus, which is one of the key targets for promoting TIAs production by genetic engineering.

Terpenoid Indole Alkaloids Biosynthesis and Metabolic Engineering in Catharanthus roseus

Catharanthus roseus L. （Madagascar periwinkle） biosynthesizes a diverse array of secondary metabolites including anticancer dimeric alkaloids （vinblastine and vincristine） and antihypertensive alkaloids （ajmalicine and serpentine）. The multi-step terpenoid indole alkaloids （TIAs） biosynthetic pathway in C. roseus is complex and is under strict molecular regulation. Many enzymes and genes involved in the TIAs biosynthesis have been studied in recent decades. Moreover, some regulatory proteins were found recently to control the production of TIAs in C. roseus. Based on mastering the rough scheme of the pathway and cloning the related genes, metabolic engineering of TIAs biosynthesis has been studied in C. roseus aiming at increasing the desired secondary metabolites in the past few years. The present article summarizes recent advances in isolation and characterization of TIAs biosynthesis genes and transcriptional regulators involved in the second metabolic control in C. roseus. Metabolic engineering applications in TIAs pathway via overexpression of these genes and regulators in C. roseus are also discussed.

Strictosidine synthase,an indispensable enzyme involved in the biosynthesis of terpenoid indole andβ-carboline alkaloids

Terpenoid indole(TIAs)andβ-carboline alkaloids(BCAs),such as suppressant reserpine,vasodilatory yohimbine,and antimalarial quinine,are natural compounds derived from strictosidine.These compounds can exert powerful pharmacological effects but be obtained from limited source in nature.the whole biosynthetic pathway of TIAs and BCAs,The Pictet–Spengler reaction catalyzed by strictosidine synthase(STR;EC:4.3.3.2)is the rate-limiting step.Therefore,it is necessary to investigate their biosynthesis pathways,especially the role of STR,and related findings will support the biosynthetic generation of natural and unnatural compounds.This review summarizes the latest studies concerning the function of STR in TIA and BCA biosynthesis,and illustrates the compounds derived from strictosidine.The substrate specificity of STR based on its structure is also summarized.Proteins that contain sixbladed four-strandedβ-propeller folds in many organisms,other than plants,are listed.The presence of these folds may lead to similar functions among organisms.The expression of STR gene can greatly influence the production of many compounds.STR is mainly applied to product various valuable drugs in plant cell suspension culture and biosynthesis in other carriers.

Enhancing terpenoid indole alkaloid production by inducible expression of mammalian Bax in Catharanthus roseus cells

Bax,a mammalian pro-apoptotic member of the Bcl-2 family,triggers hypersensitive reactions when expressed in plants.To investigate the effects of Bax on the biosynthesis of clinically important natural products in plant cells,we generate transgenic Catharanthus roseus cells overexpressing a mouse Bax protein under the β-estradiol-inducible promoter.The expression of Bax in transgenic Catharanthus roseus cells is highly dependent on β-estradiol concentrations applied.Contents of catharanthine and total terpenoid indole alkaloid of the transgenic cells treated with 30 μmol/L β-estradiol are 5.0-and 5.5-fold of the control cells.Northern and Western blotting results show that expression of mammalian Bax induces transcriptional activation of Tdc and Str,two key genes in terpenoid indole alkaloid bio-synthetic pathway of Catharanthus roseus cells,and stimulates the accumulation of defense-related protein PR1 in the cells,showing that the mouse Bax triggers the defense responses of Catharanthus roseus cells and activates the terpenoid indole alkaloid biosynthetic pathway.Thus,our data suggest that the mammalian Bax might be a potential regulatory factor for secondary metabolite biosynthesis in plant cells and imply a new secondary metabolic engineering strategy for enhancing the metabolic flux to natural products by activating the whole biosynthetic pathway rather than by engineering the single structural genes within the pathways.

Involvement of nitric oxide signaling in mammalian Bax-induced terpenoid indole alkaloid production of Catharanthus roseus cells

Bax, a mammalian pro-apoptotic member of the Bcl-2 family, has been demonstrated to be a potential regulatory factor for plant secondary metabolite biosynthesis recently. To investigate the molecular mechanism of Bax-induced secondary metabolite biosynthesis, we determined the contents of nitric oxide (NO) of the transgenic Catharanthus roseus cells overexpressing a mouse Bax protein and checked the effects of NO specific scavenger 2,4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1- oxyl-3-oxide (cPITO) on Bax-induced terpenoid indole alkaloid (TIA) production of the cells. The data showed that overexpression of the mouse Bax in C. roseus cells triggered NO generation of the cells. Treatment of cPITO not only inhibited the Bax-triggered NO burst but also suppressed the Bax-induced TIA production. The results indicated that the mouse Bax might activate the NO signaling in C. roseus cells and induce TIA production through the NO-dependent signal pathway in the cells. Furthermore, the activities of nitric oxide synthase (NOS) were significantly increased in the transgenic Bax cells as compared to those in the control cells, showing that the mouse Bax may induce NOS of C. roseus cells. Treatment of the transgenic Bax cells with NOS inhibitor PBITU blocked both Bax-induced NO genera- tion and TIA production, which suggested that the mouse Bax might trigger NO generation and TIA production through NOS. However, the NOS-like activities and NO generation in the transgenic Bax cells did not match kinetically and the Bax-induced NOS-like activity was much later and lower than NO production. Moreover, the Bax-induced NO generation and TIA production were only partially inhibited by PBITU. Thus, our results suggested that the Bax-induced NO production and secondary metabolite biosynthesis in C. roseus cells was not entirely dependent on NOS or NOS-like enzymes.

长春花萜类吲哚生物碱的生物合成途径

药用植物长春花含有130余种萜类吲哚生物碱,该文对近年来国内外有关长春花生物碱合成的上游和下游阶段及其相关研究进行详细的归纳总结。长春花上游合成途径中在相应的酶促作用下由吲哚途径产生的色胺和由类萜途径产生的裂环马钱子苷在异胡豆苷合成酶的催化作用下形成了所有长春花TIAs的共同前体物质3α-异胡豆苷,3α-异胡豆苷再由下游途径的各种酶促作用下生成种类各异的长春花TIAs。通过对长春花TIAs合成途径的阐述,为萜类吲哚生物碱合成及其代谢调控的相关研究提供参考。

长春花萜类吲哚生物碱含量测定及相关基因的表达分析

采用反相高效液相色谱法(RP-HPLC)对23个长春花品种中的重要萜类吲哚生物碱文多灵、长春质碱和长春碱含量进行了测定,发现这3种生物碱的含量在不同品种中存在较大差异。相关性分析表明,文多灵、长春质碱和长春碱这3种生物碱中,文多灵含量和长春碱含量有极显著的相关性(P<0.01)。利用RT-PCR分析了香叶醇10-脱羧酶基因(g10h)和异胡豆苷合成酶基因(str)在萜类吲哚生物碱含量有显著差异的品种之间的表达水平差异,并结合生物碱含量数据结果,发现g10h和str的表达水平和文多灵和长春质碱的总含量有显著的正相关性(P<0.05),说明g10h和str基因可以作为长春花中文多灵和长春质碱含量的参考基因标记。该研究对为选育高萜类吲哚生物碱含量长春花品种及长春花萜类吲哚生物碱代谢工程具有重要意义。

吲哚生物碱生物合成研究进展

生物碱种类繁多,吲哚类生物碱是迄今为止发现最多的一类,是许多药用植物的药效成分,具有多样的生物活性。随着代谢组学技术的发展,研究药效物质的生物合成途径对阐明植物体内代谢的分子机制以及代谢调控机理具有重要意义,对进一步研究提高药效物质的生物合成产量具有指导意义。本文按照吲哚生物碱结构进行分类,对数种吲哚生物碱的生物合成途径研究进展进行综述,以期为吲哚生物碱的生物合成机制的进一步研究奠定基础,为其他吲哚生物碱类成分的生物合成研究提供参考依据。

茉莉素介导的长春花生物碱次生代谢转录调控机制

萜类吲哚生物碱(terpeniod indole alkaloids,TIAs)是植物中产生的一类具有药理活性的次生代谢产物.药用植物长春花(Catharanthus roseus)因含有长春碱和长春新碱等重要的抗肿瘤萜类吲哚生物碱而成为研究TIAs次生代谢的主要模式植物.应用正、反向遗传学和各种代谢组学技术对长春花TIAs次生代谢途径及其调控进行了较深入的研究,相继鉴定了参与TIAs代谢途径调控的CrORCAs、CrMYCs、CrZCTs和CrWRKYs等转录因子,特别是发现茉莉素(jasmonates,JAs)介导TIAs生物合成的转录调控网络.本文以长春花TIAs生物合成途径为模式,重点论述其代谢途径中的关键酶、参与调节的转录因子,尤其是茉莉素介导的调控网络及机制,解析植物中这些天然抗癌生物碱合成积累水平低的制约因素和组织细胞特异性,讨论基于这些新知识的长春花抗肿瘤TIAs代谢工程策略和工厂化绿色生产前景.

长春花叶片三种生物碱含量及合成基因表达的初步研究

为探明长春花叶片中生物碱的动态变化和生物碱合成途径中的关键酶基因的表达量,利用高效液相色谱技术(HPLC)测定了5对真叶期、开花前期、出现花蕾期、出现开花期、成熟期1和成熟期2等6个生长阶段的长春花幼嫩、中等成熟、成熟三种叶片的生物碱(文多灵、长春质碱、长春碱)的含量。幼嫩叶片中文多灵、长春质碱和成熟叶片中长春碱的含量最高。长春花叶片的最佳采集期是花蕾期到开花期。利用荧光定量PCR技术检测了长春花叶片中生物碱合成途径中10个相关基因的相对表达量,GGPP,G10 H,SLS,STR,D4 H及DAT这6个基因对生物碱含量贡献较大,ASA及TDC基因对生物碱的增加的作用不明显。

萝芙木异胡豆苷-β-D-葡萄糖苷酶(SGD)基因的克隆与分析

[目的]对萝芙木异胡豆苷-β-D-葡萄糖苷酶(SGD)基因进行克隆与分析。[方法]以萝芙木为材料,采用RACE技术,从萝芙木中克隆SGD的全长cDNA,并对其进行表达谱分析和相关生物信息学分析。[结果]萝芙木SGD基因cDNA全长1 856 bp,包含1 608 bp的开放阅读框,编码536个氨基酸,预测分子量为61 kDa,等电点pI为6.16;生物信息学分析显示,RvSGD与蛇根木SGD及长春花SGD的序列相似性分别高达90.1%和70.9%,RvSGD也具有SGD蛋白的3个保守氨基酸催化位点His-161、Glu-207和Glu-419;荧光定量PCR表明,RvSGD在树皮中表达量最高,其次是老叶、根、嫩叶和茎。[结论]RvSGD基因的克隆和功能分析有助于在分子水平上更好地了解这个基因在TIAs的生物合成中的作用,并为今后调控TIAs的生物合成提供了新的靶点。

钩藤管家基因筛选及生物碱合成相关基因的表达分析

为研究钩藤不同部位中最适管家基因与钩藤生物碱上游合成途径中关键酶基因的表达模式。以钩藤根、茎钩、叶片、蒴果为实验材料,通过RT-qPCR技术、GeNorm、NormFinder、Bestkeeper软件及△Ct程序、RefFinder在线网站分析了12个候选管家基因(18S、SAM、TUA、TUB、EF-1β、EF-1α、RNA L13、GAPDH、Actin6、PAL、CYP、cdc73)表达稳定性。结果表明,最适管家基因为SAM。再以SAM为管家基因,基于“基因组+转录组+代谢组”共表达分析筛选出钩藤生物碱上游合成途径中的15个重点候选相关基因(G8H、8-HGO、IS、CYP76A26、7-DLGT、7-DLH、LAMT、SLS、AS、AnPRT、IGPS、TSA、TSB、TDC、STR)进行表达分析,结果显示这些基因的表达量与含量趋势较为一致,很可能参与钩藤中TIAs的合成。

Cloning and Characterization of the Strictosidine-β-D-glucosidase (SGD) Gene from Rauvolfia verticillata

[Objective] This study aimed to clone Scrictosidine-β-D-glucosidase (SGD) gene from Rauvolfia verticillata and analyze its characteristics. [Method] The full-length cDNA of SGD was cloned from R. verticillata with RACE technique. Then the expression levels in different tissues were analyzed with quantitative RT-PCR and the bioinformatic characteristics were also predicted. [Result] The full-length cDNA of RvSGD was 1 856 bp, containing a 1 608 bp CDS that encoded 536 amino acids with a calculated molecular mass of 61.0 kDa and an isoelectric point of 6.16. Bioinformatic analysis revealed that RvSGD shared high similarity with SGDs from Cantharanthus roseus and Rauvolfia serpentina at the amino acids; three conserved catalytic sites His-161, Glu-207 and Glu-419 were also presented in RvSGD. Quantitative RT-PCR showed that expression level of RvSGD was the highest in barks, followed by old leaves, roots, tender leaves and tender stems. [Conclusion] The present study helps to understand more about the functions of the SGD gene at the level of molecular genetics, and provides new targets for molecular regulation of TIAs biosynthesis.

外源脱落酸对钩藤中生物碱合成的影响

为了探究外施ABA对钩藤中活性成分钩藤碱(RIN)和异钩藤碱(IRN)合成与积累以及生物碱合成途径基因表达的影响,以期进一步揭示RIN和IRN生物合成对ABA的响应机制。本研究以6月龄的钩藤为材料,外施不同浓度的ABA,利用HPLC测定不同浓度脱落酸处理后钩藤叶中RIN和IRN的含量积累情况,采用荧光定量PCR (RT-qPCR)分析合成途径中关键酶基因STR、TDC、SGD和G8H等基因的表达。结果表明,100μmol/L的ABA溶液最适于促进钩藤叶中生物碱的合成积累,处理后2 d两种生物碱含量就比对照提高了1倍;同时,q PCR显示合成途径中SGD、TDC、G8H和STR家族酶基因均有不同程度的上调。因此,合理外施ABA溶液有助于促进钩藤活性成分的合成与积累,提高钩藤的经济效益,对钩藤药材的高效优质种植管理提供了理论依据,也为阐明钩藤生物碱生物合成的分子调控机制提供了基础。

药用植物长春花WRKY转录因子的鉴定及表达谱分析

WRKY是调控植物生长发育和逆境胁迫反应等生命活动的一个转录因子大家族。然而,有关药用植物长春花CrWRKY转录因子的种类和功能却知之甚少。从26 009个长春花蛋白中鉴定出47个CrWRKY转录因子。依据WRKY结构域和系统进化,将CrWRKY分为G1、G2和G3三大类群。表达谱数据分析表明,长春花CrWRKY基因的表达具有器官特异性。47个CrWRKY基因的表达谱可分为3种表达模式:第1类型主要在花、甲基茉莉酸甲酯(MeJA)或酵母提取物(YE)处理的原生质体中高表达;第2类型主要在茎和毛状根中高表达;第3类型在根、茎、叶、幼苗和MeJA处理的毛状根中高表达。进一步用实时定量PCR检测了16个代表性CrWRKY基因在不同器官、MeJA处理原生质体和毛状根中的表达模式,检测结果与上述数字基因表达谱数据基本一致。约1/3以上CrWRKY基因的表达受MeJA和YE的调控,暗示它们可能参与萜类吲哚生物碱的合成和逆境胁迫反应。为进一步解析长春花WRKY转录因子的功能和萜类吲哚生物碱合成调控的网络奠定了基础。

长春花密码子使用偏好性分析

以长春花为研究对象,分析其密码子使用偏好性,以期为相关基因的异源表达、基因的预测、物种的进化研究提供指导。该研究以长春花的30 437条蛋白质编码序列为数据来源,对长春花密码子组成和密码子偏性的各项参数进行了计算和统计分析。计算了长春花萜类吲哚生物碱(terpenoid indole alkaloids,TIAs)生物合成途径中25个关键酶基因含有大肠杆菌或酿酒酵母稀有密码子的比例。结果显示,长春花基因的平均GC量为42.47%,密码子第3位碱基平均GC量为35.89%。长春花中共有28个密码子的同义密码子相对使用度(relative synonymous codon usage,RSCU)大于1,其中26个以A或T结尾。25个关键酶基因含有大肠杆菌稀有密码子的比例明显高于酿酒酵母稀有密码子的比例。长春花主要偏爱使用以A和T结尾的密码子;相比于酿酒酵母,其密码子使用特点与大肠杆菌的差异更大,推测酿酒酵母可能是长春花基因更合适的异源表达宿主。

拟南芥基因组中注释为异胡豆苷合成酶的基因克隆及异源表达

拟南芥(Arabidopsis thaliana)是生物学研究中广泛使用的模式生物.到目前为止,没有证据表明拟南芥中存在复杂生物碱,但对其基因组数据的生物信息学分析表明,有很多基因与萜类吲哚生物碱(TIAs)生物合成途径中的相关基因有高度的同源性.为了从分子水平研究拟南芥是否具有合成该类复杂生物碱的潜力,首先对拟南芥中可能编码异胡豆苷合成酶(STR)的15个基因及其编码的酶进行广泛的生物信息学分析和计算;在此基础上,选择其中5个AtSTR基因为目的基因,设计特异性引物;从拟南芥中提取总RNA,一步法RT-PCR克隆得到上述基因;通过TA克隆将上述基因转入pGM-T载体,筛选、酶切及PCR鉴定,DNA测序验证它们的核酸序列;将测序验证的AtSTR基因经过PCR扩增、质粒构建、转入Escherichia coli BL21(DE3)中进行异源表达和蛋白纯化;在整体细胞、细胞裂解液、细胞裂解液上清和纯化蛋白共4个层次进行AtSTR酶功能表征,未发现目标产物或新产物的生成;上述基因的具体功能还有待进一步研究.

长春花萜类吲哚生物碱生物合成与调控研究

长春花含有多种具有重要药理活性的萜类吲哚生物碱(TIA)。TIA的生物合成与调控及其合成生物学研究受到广泛关注。3α(S)-异胡豆苷是TIA生物合成的重要节点,由裂环马钱子苷和色胺缩合而成。前者通过环烯醚萜途径生成;后者通过吲哚途径生成。由3α(S)-异胡豆苷分别经过多步酶促反应生成文多灵和长春质碱,然后两者缩合生成α-3,4-脱水长春碱,进而生成长春碱和长春新碱。AP2/ERF和WRKY等多种转录因子参与了TIA合成的调控。长春花TIA生物合成途径的逐步解析为其合成生物学研究奠定了基础。目前已在酿酒酵母实现了3α(S)-异胡豆苷和文多灵等的异源合成。长春花TIA生物合成与调控的研究将为TIA类药物的生产和研发提供支撑。

长春花吲哚萜类生物碱代谢途径研究进展

长春花(Catharanthus roseus(L.)G.Don.)含有130余种生物碱,既是抗癌药物长春碱和长春新碱的唯一植物来源,也是研究吲哚萜类生物碱代谢途径的模式植物。生成长春花生物碱的前体环烯醚萜主要来源于2-C-甲基-D-赤藓醇4-磷酸途径(MEP),甲羟戊酸途径(MVA)可能在此过程中起辅助作用。吲哚生物碱代谢途径受茉莉酸甲酯正调控。文多灵途径过去被认为在长春花悬浮细胞中没有文多灵产生,而目前的研究发现文多灵在长春花C20hi细胞中可以微量合成。文章对近年来长春花吲哚生物碱代谢途径及部分关键酶研究进展进行了综述。

钩吻看家基因筛选及生物碱合成相关酶基因的表达分析

钩吻是我国的一种传统中草药,具有重要的药用价值,萜类吲哚生物碱为其主要的活性组分。为了研究钩吻不同部位最适看家基因,以钩吻4个不同部位根皮、茎段、叶片和花序为材料,通过实时荧光定量PCR技术(quantitative real-time PCR,qRT-PCR),以及GeNorm、NormFinder、BestKeeper、ΔCT和RefFinder软件对10个候选看家基因(18S、GAPDH、Actin、TUA、TUB、SAND、EF-1α、UBC、UBQ和cdc25)进行表达稳定性评估。结果显示,EF-1α在钩吻的4个部位中均稳定表达,为最适的看家基因。同时,为了研究与生物碱合成相关基因的表达情况,基于“基因组+全长转录组+代谢组”共表达模式筛选出钩吻生物碱合成通路上18个相关基因(AS、AnPRT、PRAI、IGPS、TSA、TSB、TDC、GES、G8H、8-HGO、IS、7-DLS、7-DLGT、7-DLH、LAMT、SLS、STR和SGD),以看家基因EF-1α为参照,利用qRT-PCR技术对这18个候选基因表达情况进行分析,结果发现这些基因的表达量与钩吻素己含量变化呈相关性,推测其可能参与钩吻生物碱钩吻素己的合成。