【作者】 戴怡龄；

【导师】 唐克轩；

【作者基本信息】 复旦大学 ， 遗传学， 2008， 博士

【摘要】 异戊二烯代谢途径（Isoprenoid Biosynthetic Pathway）是植物萜类次生代谢物合成的重要途径,该途径中生成的异戊二烯类代谢产物包括青蒿素、紫杉醇、银杏内酯和其他萜类吲哚生物碱等都具有重要的经济价值。其中,红豆杉（Taxuscuspidata）异戊二烯代谢途径产物之一的紫杉醇(Taxol^?)因其独特的抗癌机理和广谱性,在临床上被广泛应用,但受红豆杉生长缓慢、紫杉醇含量低和合成成本高等因素的制约,其产量一直难于提高,使得运用基因工程手段来提高紫杉醇生物合成的研究成为近年来次生代谢工程中的研究热点之一。转录因子（Transcription Factor,TF）作为后基因组中改造植物代谢途径的一种新型工具,具有从宏观调控次生代谢通量的优势。目前与次生代谢途径相关的转录因子研究多集中在苯丙氨酸代谢途径和生物碱代谢途径上,已报道的与萜类合成转录水平调控相关的转录因子只有TIA合成途径中的ORCA1-3家族和ZCT1-3家族,而对于红豆杉中异戊二烯代谢途径相关转录因子的研究还处于空白。本文首次利用酵母单杂交（Yeast One-Hybrid）方法和cDNA末端快速扩增技术（Rapid-Amplification of cDNA Ends,RACE）从东北红豆杉cDNA文库中克隆到两个AP2类转录因子基因,TcAP2和TcDREB,并对它们与红豆杉异戊二烯代谢途径紫杉醇合成途径中的多个关键酶基因的启动子的结合情况进行了研究,为深入研究AP2类转录因子能否参与对紫杉醇合成的转录调控奠定了基础。以JERE作诱饵,利用酵母单杂交的建库和筛选方法首次从东北红豆杉中克隆到与MeJA诱导相关的AP2类转录因子基因TcAP2,其开放阅读框（OpenReading Frame,ORF）编码长度为268个氨基酸的蛋白,分子量29.6kD,等电点5.05,拓扑结构显示该转录因子具有典型的AP2结构域特征:3个反向平行的β-折叠和1个α-螺旋。其DNA结合域内有DREB亚族特有的Val¹⁴和Ala³⁷氨基酸残基,分别是DREB亚族转录因子与DRE元件和GCC Box元件的结合位点。进化树分析及同源比对分析都表明,该蛋白与拟南芥（Arabidopsis thaliana）中的At1g21910基因所编码的AtAP2和小立碗藓（Physcomitrella patens）中的PpAP2转录因子进化关系最近,属于同一分支,其DNA结合域的一致性达到85%以上。通过Southern blot分析表明该基因在东北红豆杉中以单拷贝形式存在。通过RACE方法从东北红豆杉中克隆到第二个AP2类转录因子基因TcDREB,其ORF编码410个氨基酸的蛋白,也具有典型的AP2结构域,空间构象及结合的顺式元件类型与TcAP2类似。进化树分析和多重比对表明,就DNA结合域而言,该转录因子与长春花（Catharanthus roseus）的ORCA1和大豆（Glycinemax）的GmDREBa的一致性最高。Southern blot表明该基因在东北红豆杉中也以单拷贝形式存在。亚细胞定位结果表明两个转录因子都定位在细胞核中。半定量RT-PCR分析显示,它们在幼茎中的表达量最高。RT-QPCR（Real-time Quantitative PCR）表明TcAP2转录水平的表达主要受MeJA和SA、高盐和低温的诱导;而TcDREB的表达受高盐、MeJA和SA以及ABA的诱导。凝胶电泳迁移率实验（Electrophoretic Mobility Shift Assays,EMSA）显示,两个转录因子都能与DRE顺式元件结合。其中TcDREB还能与紫杉醇合成途径中的TS、5α、10β和13α四个关键酶基因的启动子以及TS基因启动子上的GCC Box结合,而突变后的GCCBox不结合TcDREB。进一步用原子力显微镜（Amotic Force Microscope,AFM）证实其结合的特异性,发现加入游离的TcDREB蛋白能使其与DRE元件的结合概率从（19.3±5.2）%下降到（3.9±1.3）,而与MDRE的结合概率仅为（4.5±1.6）%,结合成键分布概率验证了TcDREB与DRE元件及四个启动子结合的特异性。综合分析认为,TcDREB与红豆杉异戊二烯代谢产物紫杉醇的合成途径相关,并且这种相关性可能是受ABA信号传导途径调控的。对于TcDREB结合启动子上的顺式元件的具体类型还有待进一步启动子缺失的结合实验证明。为了探明这两个转录因子在红豆杉中与异戊二烯代谢途径的产物紫杉醇的合成途径的相关性,进一步构建了TcAP2基因和TcDREB基因的植物表达载体并转化拟南芥;同时构建了TS、5α、10β和13α四个关键酶基因的启动子融合gus基因的植物表达载体与转录因子基因植物表达载体共转化拟南芥,已获得转TcDREB基因的阳性植株,为进一步研究其在紫杉醇代谢中的作用打下了基础。更多还原

【Abstract】 Isoprenoid biosynthetic pathway plays an important role in plant terpenoid natural metabolism pathways and terpenoids’ synthesis.Most of the products (Artemisinin,Taxol,Ginkgolide) in isoprenoid biosynthetic pathway have been broadly applied in medical field and clinical therapy for human beings.Therefore, studying the isoprenoid biosynthetic pathway in Taxus cuspidata is important for further understanding the regulation and biosynthesis of taxol,the important anti-cancer chemical produced in Taxus trees.Taxus is an ancient and precious species.Taxol(paclitaxel) is one of natural diterpenoid alkaloids firstly isolated from the bark of the yew(Taxus brevifolia). Taxol has been well established and approved by FDA as a very important effective chemotherapeutic agent against a wide range of tumors since 1992.However,the supply of Taxol has been limited since the discovery of this natural product,and with increasing applications in chemotherapy,the availability and cost of the drug will remain an important issue.Transcription factors(TFs),found to be regulators of metabolic flux,have become a powerful tool for the manipulation of complex metabolic engineering in plants.In this thesis,two novel AP2 TF genes,TcAP2 and TcDREB,which may be involved in taxol biosynthetic pathway,has been isolated from Taxus cuspidata for the first time.Probing into the induction effects of methyl jasmonate(MeJA) and salicylic acid (SA) to taxol content,suspension cells of T.cuspidate were treated with exogenous MeJA and SA before construction of yeast one-hybrid library.Based on spatial distribution frequency of C-13 phenylpropanoid side chain-CoA acyltransferase (BAPT),we explored the optimal induction time of MeJA and SA to the suspension cells for the construction of cDNA library.The bait plasmid was constructed with cis-element JERE and 72 positive plasmids were selected from T.cuspidata cDNA library by using yeast one-hybrid system.A cDNA-coded TF encoding 268 amino acids was obtained.It had a conserved AP2/EREBP domain in which the N-terminal had a 4-amino acid nuclear localization signal(NLS) and C-terminal had a transcriptional activation domain, named as TcAP2(GenBank accession number EU549860).Multiple alignments and phylogenetic tree analysis indicated that TcAP2 was a member of AP2/EREBP transcription factor gene family and the deduced TcAP2 protein had the highest homology with At1g21910 from Arabidopsis thaliana and PpAP2 from Physcomitrella patens,which possibly reflected the similar functions among them. The binding site V14 inβ-sheet of YRG element and A37 in or-helix of RAYD element was conserved in AP2/EREBP domain which reflected its binding ability to DRE cis-element and GCC Box.Via SMART Rapid Amplification of 5’ and 3’-cDNA Ends(RACE),another novel AP2 transcription factor gene,TcDREB(GenBank accession number EU549861),was cloned from T.cuspidata.The full-length cDNA of TcDREB contained a 1233-bp ORF,encoding 410 amino acids with no N-terminal propeptide. Predicted tertiary structure showed it had the conserved AP2 domain like TcAP2. Multiple alignments showed TcDREB was also a member of the DREB subfamily. TcDREB also possessed threeβ-sheets and oneα-helix as well as V14 and A37 DNA binding sites.Subcellular location analysis of TcAP2 and TcDREB demonstrated that they were nuclear-localized proteins.Semi-quantitative RT-PCR revealed that TcAP2 and TcDREB constitutively expressed in all test tissues,including stems,roots,and leaves, with high expression in new stems.EMSA of TcAP2 and TcDREB analysis indicated that both of them could bind to DRE element.Besides,TcDREB could bind to the promoters of 10β,13α,5αand TS as well as GCC Box of TS promoter in taxol biosynthesis pathway but not to MGCC Box.Determination of binding specificity was testified by AFM force measurement. The mean value of single molecular interaction force of TcDREB-DRE was determined as(76.3±2.3) pN.The binding probability was(19.3±5.2).The probabilities of TcDREB and other four promoters were ranged from 15%to 20%, suggesting the regulation of TcDREB in taxol biosynthesis pathway of T.cuspidata.The expression of TcAP2 and TcDREB could be induced by MeJA,low temperature and salt,ABA and salt,suggesting their involvement in isoprenoid biosynthetic pathway and ABA-dependent signal transduction.In order to analyze the regulation of the two transcription factors in isoprenoid biosynthetic pathway,the over-expression vector containing TcAP2 or TcDREB cDNAs driven by the CaMV 35S promoters and vectors carrying TS/5α/10β/13α-promoter-GUSA were constructed and single or co-transformed into A. thaliana.Transgenic plants transformed with TcDREB over-expression vector were generated,which will be used to test the effects of stress on electrolyte leakage rate in the transgenic plants in further study.In summary,the cloning and analysis of TcDREB and promoters of 10β,13α,5αand TS provide the basis for further studying the detailed role of TcDREB in regulation of taxol biosynthetic pathway of T.cuspidata.更多还原