【作者】 杨瑞仪；

【导师】 陈沛泉；

【作者基本信息】 广州中医药大学 ， 中医临床基础， 2009， 博士

【摘要】 植物次生代谢产物是医药、化工、食品和农业高附加值产品的重要原料,其中萜类化合物是植物次生代谢产物的主要组成部分。抗疟药青蒿素及抗肿瘤药紫杉醇、长春花碱等都是著名的萜类药物,其碳链骨架都是通过细胞质或质体的异戊二烯途径合成的。目前,萜类生物合成途径的中间产物及其转变过程已基本阐明,但对萜类合成中基因表达调节的分子机制却了解甚少。为了建立萜类合成基因表达研究平台,本研究以菊科蒿属植物青蒿为模式,采用RTFQ-PCR技术及GUS报告基因表型显示工具,系统而全面地开展青蒿素及其相关萜类生物合成基因在天然及人工模拟环境中的转录调控机制研究。同时,在前期工作基础上,提出“¹O₂可能是青蒿素合成诱导剂”的假说,并通过实验予以证实。本研究的目的在于建立萜类合成代谢共同途径研究模型;构筑青蒿倍半萜青蒿素合成基因表达研究平台;阐明青蒿素合成诱导及时空调节机制;探索青蒿素高产的可行方法,其主要研究内容及其成果概括如下:①从青蒿中克隆了8种MVA及DXP途径基因（HMGR、FPS、DBR2、DXS、DXR、FS、CS、EPS）并测得全序列,其中DBR2基因序列为首次发表,已提交GenBank注册（登录号为EU848577）,连同我们前期已克隆的ADS、CYP71AV1、CPR、SQS基因,为进一步开展青蒿功能基因组学研究及青蒿素高产代谢基因工程改良提供了丰富的素材。②采用自行设计的扩增引物,对青蒿中9种MVA及DXP途径基因（HMGR、FPS、ADS、CYP71AV1、CPR、DBR2、DXS、DXR、SQS）的发育特异表达动态进行了跟踪测定,发现相应的mRNA在青蒿中的水平以6月份相对较低,7月份后迅速提高,到8月份（临近开花前）达到顶峰,9月份（开花后）开始回落,其中ADS与CYP71AV1mRNA水平在7月份后更是出现爆发式增长,最高峰时分别为6月份的40倍和18倍。同时还发现,MVA及DXP途径的其他基因与青蒿素合成基因的发育表达模式基本一致,间接表明细胞质MVA途径及质体DXP途径可能共同参与青蒿素的生物合成。③在青蒿组织特异表达动态方面,处于盛花期的青蒿根、茎、叶、花中都能检测到ADS、CYP71AV1、CPR、DBR2等青蒿素合成基因的表达,而且各基因表达量差异不显著,表明苗期至盛花期间的青蒿素合成基因表达无明显组织特异性。相反,在褐变的老叶中,青蒿素合成相关基因mRNA水平显著提高,其中CYP71AV1、ADS、DBR2mRNA的上升幅度最明显,分别是绿叶的51.2、25.5、14.8倍。相应地,老叶中的青蒿素含量也相应提高,最高约为绿叶的1.5倍,从而首次发现青蒿素合成基因表达与青蒿素的积累可能受生长发育程序尤其是衰老进程的直接调控。由于老叶比绿叶释放更多的¹O₂,推测¹O₂直接参与了青蒿素合成基因表达的调控及青蒿素前体向青蒿素的非酶促转变,因而它可能是衰老过程能促进青蒿素大量合成与积累的主要原因。④在转基因烟草ADSP-GUS融合系统中,根、茎、叶中都能检测到GUS活性,表明ADS启动子驱动GUS报告基因的基础表达,在转录调控上无明显组织特异性。GUS定量检测结果显示,在低温（4℃）及紫外辐射条件下,GUS活性分别提高2.2倍和1.6倍,提示ADS基因受极端环境胁迫的诱导调控,对诱导¹O₂生成的胁迫条件（如低温、紫外辐射等）高度响应。逆境诱导报告基因表型显示平台的成功建立,为进一步高通量筛选青蒿素合成基因高效表达诱导剂提供了极大方便。⑤利用RTFQ-PCR技术分析了青蒿素生物合成相关基因在低温（4℃）、高温（50℃）、紫外辐射、淹水或复水（缺氧）、脱水（干旱）、NaCl（高盐）、真菌诱激（添加酵母提取物）、信号分子刺激（SA、MJ、GA₃、ABA）等12种诱生¹O₂的人工模拟环境中的转录模式。结果显示,在低温与紫外辐射条件下,青蒿素生物合成途径（包括共同途径与特异途径）的基因表达水平普遍上升;ADS和DBR2对于环境胁迫的响应更为明显:ADS在冷、紫外辐射、脱水、添加酵母提取物处理条件下的转录水平分别是对照的35.6、14.2、15.5、5.5倍,DBR2在淹水、复水、添加酵母提取物处理条件下的转录水平分别是对照的1 3.8、38.2和20.7倍。由于上述氧化胁迫条件均能导致¹O₂释放,因而本研究在成功模拟包括天然衰老过程在内的氧化胁迫条件的基础上,首次揭示了青蒿素合成基因受氧化胁迫尤其是¹O₂诱导的分子机理。⑥对本实验室首次克隆的7种青蒿新EST——过氧化物酶1基因（POD1）、几丁质酶基因（CH1）、钙调素基因（CaM）、泛素结合酶基因（UCE）、干旱/低温及盐响应蛋白基因（D/LTSRP）、富含甘氨酸RNA结合蛋白基因（RGP）和生长素阻遏/休眠相关蛋白基因（AR/DAP）的低温诱导表达模式进行了定量分析。结果显示,经过4℃处理48h以后,D/LTSRP、UCE、CaM、AR/DAP、POD1基因的mRNA水平分别为对照的7.5、5.0、2.6、2.3、1.5倍,提示这些基因可能参与了青蒿低温信号启动与转导的网络调控,从而为进一步阐明青蒿素生物合成的信号转导通路及其级联调节网络提供了线索。⑦对细胞质与质体萜类合成途径受到抑制后的基因表达定量分析显示,MVA与DXP两条途径表现互为补偿的机制,添加DXP途径抑制剂（磷甘霉素）后,DXP途径基因表达出现先抑制后恢复,而MVA途径基因表达则呈现先上升后回落的趋势;添加MVA途径抑制剂（洛伐他汀）后,两条途径的基因表达均被抑制,进一步表明不同的亚细胞空间（细胞质和质体）在萜类合成过程中存在着交叉对话,再次证实MVA与DXP途径都参与了青蒿素的生物合成。咪康唑对类固醇合成的抑制显示,青蒿素合成基因ADS和DBR2的表达分别提高了2倍和5倍,表明对青蒿素合成竞争途径的抑制可调节MVA途径不同分支中碳源的流向,使细胞代谢向合成青蒿素的方向移动。⑧对转asSQS基因青蒿株中SQS基因及其他倍半萜合酶基因的表达变化研究表明,在转基因株中,SQS基因的表达受到明显抑制,为非转基因的0.5-0.12倍,有2株转基因株中的ADS基因表达提高3.5和3倍。经过4℃间歇冷处理后,非转基因与转基因株中的SQS基因表达不受影响,ADS与EPS基因的表达水平上调1.5-5倍,FS基因表达基本维持不变,CS基因的表达量下降,下降幅度约为50-100倍。再次证明青蒿中萜类合成具有网络调控特性,在“节流”与“引流”的双重作用下更有利于吸引碳源流向青蒿素合成途径。本研究的创新性在于首次从转录水平上开展了包括青蒿素合成基因在内的青蒿萜类代谢调控研究,为其他萜类（如紫杉醇、长春花碱等）的后续功能基因组学及代谢途径工程研究提供了模式,有关青蒿萜类基因表达及其调控的研究结果国内外均无报道。其次,通过对青蒿素合成基因的时间及空间表达动态的跟踪测定,初步揭示了青蒿素合成基因表达的发育及组织特异调节机制,首次发现青蒿素合成基因表达受衰老程序控制的规律,证实衰老过程中产生的过量¹O₂可能是诱导青蒿素合成基因高效表达及促进青蒿素高产的主要原因。在此基础上,利用各种环境胁迫条件模拟^O₂的释放,阐明了青蒿素合成基因的环境诱导表达及其调节机制,从而经由实验直接验证了“¹O₂是青蒿素合成诱导剂”的假说,并进一步指明青蒿素可能是其前体（双氢青蒿酸）清除¹O₂后生成的副产品。更多还原

【Abstract】 As important raw meterials in medicine,food,chemical industry and agriculture,plant secondary metabolites possess high commercial values.Terpenoids,such as anti-malarial agent artemisinin and anti-tumor drug taxol,constitute the most major components of plant secondary metabolites.It was widely accepted that the formation of terpenoids with a common structural five-carbon unit occur via two distinctive pathways in all plants:the mevalonate（MVA） pathway in the cytosol and the 1-deoxy-D-xylulose-5-phosphate（DXP） pathway in plastids.Until now,however,we have only known little about the molecular mechanism underlying the regulated gene expression during terpenoid biosynthesis, although most of intermediates on the terpenoid biosynthesistic pathways and their conversion processes have been typically elucidated.In order to establish a platform for investigating the expression patterns of terpenoid biosynthetic genes,Artemisia annua L,a Compositae Artemesia plant,was used as a model to systematically and comprehensively study the transcriptional regulation on the biosynthesis of artemisinin and other relative terpenoids in the natural and artificial environments by RTFQ-PCR and phenotyping the GUS reporter.Meanwhile,based on our previous work,a hypothesis proposing that singlet oxygen（¹O₂） maybe an inductor for artemisinin biosynthesis was confirmed experimentally.This paper is aimed to set up a model to disclose the common anabolism pathway of terpenoids and construct a platform to reveal the expression mode of artemisinin biosynthesis genes,to elucidate the mechanism of conditioned stimulants,temporal and spatial modulators for artemisinin production,and finally to find out feasible approaches to enhanced artemisinin production.The main topics and results of this paper are as follows:Eight MVA and DXP pathway genes（HMGR,FPS,DBR2,DXS,DXR,FS,CS and EPS） were cloned from A.annua and sequenced,among which the sequence ofDBR2 gene was summitted to GenBank for the first time（accession No.EU848577）.With inclusion of ADS,CYP71AV1,CPR and SQS genes previously cloned,there were 12 genes that encode the terpenoids biosynthetic key enzymes in A.annua available for further researches of functional genomics and metabolic engineering aming at high-yield artemisinin.To unravel the diverse expression patterns of terpenoid biosynthetic genes during the developmental stage in A.annua,the transcriptional profiles of nine MVA and DXP pathway genes（HMGR,FPS,ADS,CYP71AV1,CPR,DBR2,DXS,DXR and SQS） were quantitatively assayed by RTFQ-PCR.As results,the expression levels of all tested genes were extremely low in June,and raised dramatically in July,and reached their peak values before flowering（in August）,but dropped gradually after blooming（in September）. Especially,the great elevation of ADS and CYP71AV1 mRNA levels were detected in July, which were 40 and 18 times higher than those in June.The developmental expression pattems of other genes on the DXP pathway were consistent with those of artemisinin biosynthetic genes on the MVA pathway.Therefore,these results suggested that artemisinin was probably biosynthesized via both pathways.In the aspect of tissue-specific gene expression patterns in A.annua,artemisinin biosynthetic genes,comprising ASD,CYP71AV1,CPR and DBR2,illustrated no difference in roots,stems,leaves and flowers during flowering.Such result revealed that artemisinin biosynthetic genes in those tissues showed no obvious tissue specificity.However, artemisinin biosynthetic mRNA levels significantly elevated in senescent leaves that had tured brown.The increments of CYP71AV1,ADS,DBR2 mRNA levels were 51.2,25.5 and 14.8 times higher than those in green leaves.Accordingly,artemisinin content in senescent leaves was also promoted to 1.5 times higher than that in green leaves.In conclusion,the fashions regarding expression of artemisinin biosynthetic genes and accumulation of artemisinin might be directly regulated by the growth and developmental procedures of plants themselves,especially by the senescent process.Because senescent leaves emitted more amounts of ¹O₂ than green leaves,it is speculated that ²O₂ may be directly involved in the regulation of artemisinin biosynthetic genes and non-enzymatic conversion from artemisinin precursors to artemisinin,thereby playing a major role in facilitating artemisinin biosynthesis and accumulation.To further compile evidence on the modulatory mechanism of artemisinin biosynthesis, transgenic tobacco（Nicotiana tabacum） plants that introduced an ADSP-GUS fusion gene were cultivated in the present study and showed a typical GUS phanotype in whole plants, indicating that a basal level of ADSP-driven GUS expression didn’t exhibit tissue specificity.The quantitative results showed that the GUS activity in transgenic tobacco plants treated by low-temperature（4℃） and ultraviolet irradiation were 2.2 and 1.6 times higher than that in the control.It is suggested that the ADS gene is induced by adverse environmental stresses at least including cold and irradiation,and may be more sensitive to ¹O₂ generated by a variety of stress conditions.The successful establishment of the stress-inducible GUS reporter phenotyping system provided convenience for future high-throughput screening of inducers that stimulate over-expression of artemisinin biosynthetic genes.The transcriptional patterns of artemisinin biosynthetic genes under the circumstances of 12 artificial simulations that enhance generation of ¹O₂ were monitored by using RTFQ-PCR technology.The artificial simulative elements included low-temperature（4℃）, high-temperature（50℃）,ultraviolet irradiation,hypoxia（flooding and rehydration）, drought（dehydration）,high salt（NaCl）,fungal elicitor（yeast extract） and plant signal molecules（SA,MJ,GA₃ and ABA）.The results showed that the expression levels of artemisinin biosynthetic genes（including those on the common and specific biosynthetic pathways） generally up-regulated,in which ADS and DBR2 genes were more sensitive than others to those stimuli.The ADS transcript level in A.annua plantlets treated with cold, ultraviolet,dehydration and yeast extract were 35.6,14.2,15.5 and 5.5 times higher than that in the control,and the DBR2 mRNA level upon the treatment by flooding,rehydration and yeast extract were 13.8,38.2 and 20.7 times higher than that in the control.It is revealed for the first time that artemisinin biosynthetic genes are highly induced by oxidative stresses,especially by ¹O₂,based on the simulation outcomes under the oxidative stress conditions,including the natural senescence process,which enables the induction of sensitive genes by ¹O₂ release.Low temperature-induced expression of previously identified expressed sequence tags （ESTs） by our group were quantitatively analyzed,which included seven sequences,i.e., peroxidase 1 gene（POD1）,chitinase gene（CH1）,calmodulin gene（CaM）, ubiquitin-conjugating enzyme gene（UCE）,drought/low temperature and salt responsive protein gene（D/LTSRP）,RNA-binding glycine rich protein gene（RGP）,and auxin-repressed/dormancy-associated protein gene（AR/DAP）.The quantitative results showed that D/LTSRP,UCE,CaM,AR/DAP and POD1 mRNA levels after 4℃treatment for 48h were 7.5,5.0,2.6,2.3 and 1.5 times higher than that of the control,suggesting that these genes may be involve in the regulation throughout the metabolic networks that relevant to initiation and transduction of the low-temperature signal in A.annua.This possibility provided a clue to reveal the mechanism underlying the signal transduction pathway and cascade regulation network for artemisinin biosynthesis.A specific MVA pathway inhibitor,lovastatin and a specific DXP pathway inhibitor, fosmidomycin,were used to perturb the biosynthetic flux in A.annua seedlings.The quantitative analysis at the transcriptional level showed that a mutual compensation mechanism was present in both pathways.Treatment of seedlings with fosmidomycin resulted in a transient decline of mRNA levels on the DXP pathway.After the initial drop, the mRNA levels of DXP pathway genes recovered to those corresponding to control levels, whereas mRNA levels of MVA pathway genes first increased but then decreased.As a response to lovastatin exposure,the expression activities of both pathway genes were inhibited,further suggesting that crosstalk between cytosolic and plastidial pathways for terpenoid biosynthesis might persist,and that both MVA and DXP pathways were likely involved in artemisinin production.After supplement with miconazole,an inhibitor of sterols biosynthesis,the levels of ADS and DBR2 mRNAs increased two and five times compared to the control,suggesting that carbon source was partially shifted from sterols to sesquiterpenes.The expression patterns of squalene synthase gene（SQS） and other sesquiterpene synthase genes（ADS,CS,FS and EPS） in transgenic A.annua plants that were integrated with one or more copies of anti-sense squalene synthase genes（asSQS） were surveyed.The results showed that the SQS gene in transgenic plantlets was significantly inhibited by the generation of asSQS mRNA,and decreased about two to six times compared to the wild-type control,whereas ADS mRNA in two of transgenic plantlets increased 3.5 and 3 times.After intermittent cold-treatment,SQS and FS mRNAs exhibited no changes in transgenic and wild-type plants,whereas ADS and EPS mRNA increased 1.5 to 5 times,and CS mRNA dramatically decreased about 50-100 folds.The main innovations of this paper were summarized as follows:it is the first time to survey the expression profile of metabolic regulation on terpenoid biosynthetic genes including artemisinin biosynthetic genes at the transcription level.It provided a model for the follow-up studies on the functional genomics and metabolic engineering of other terpenoids like taxol and vinblastine.Secondly,through monitoring the expression kinetics of these genes in different subcellular spaces and during different development stages, present study has preliminarily revealed a unique mode of developmental and tissuesspecific regulation on artemisinin biosynthesis,and first discovered artemisinin biosynthesis being modulated by the senescence process.On such basis,using various environmental stress conditions that simulate the senescence process,this research has elucidated that artemisinin biosynthetic genes were inducible by various environmental stresses.It was confirmed that excessive release of ¹O₂ during the senescence process is the main cause to increase the expression of artemisinin biosynthetic genes and enhance the production of artemisinin.It was also proved the hypothesis that ¹O₂ is mostly the inducer for artemisinin biosynthesis,and further clarified that artemisinin may be a by-product generated from its precursor dihydroartemisinic acid in the process of ¹O₂ scavenging.更多还原