核基质结合区对转爬山虎芪合酶基因烟草中产生白藜芦醇的作用

采用核基质结合区(MARs)来提高转芪合酶基因(STS)烟草(Nicotianatabacum L.)中白藜芦醇产物的含量.MARs是细胞中能与核基质特异紧密结合的DNA片段,体外结合实验表明克隆自酵母的MARs序列能特异地与烟草核基质结合.芪合酶是白藜芦醇生物合成中的关键酶,用RT-PCR方法从川鄂爬山虎(Parthenocissus henryana(Hemsl.)Diels et Gilg)中克隆了与葡萄芪合酶基因有较高同源性的芪合酶编码区,将其置于CaMV35SΩ强启动子下,分别构建两侧带有MARs及不含MARs序列的表达载体,通过农杆菌介导转化烟草.Northern blot及HPLC等分析表明STS基因已整合至烟草染色体中并正常转录,且表达的外源芪合酶在烟草中可催化其底物合成白藜芦醇产物.与对照相比,MARs的存在使转芪合酶基因烟草中白藜芦醇的含量平均提高了约一倍.MARs在转芪合酶基因植物中的应用也为获得抗病性更强、白藜芦醇含量更高、更保健的转基因果蔬的研究奠定了基础.     

烟草Nt4CL与虎杖PcSTS基因的融合表达与功能分析

4-香豆酰辅酶A连接酶(4-coumarate-Co A ligase,4CL)和芪合酶(stilbene synthase,STS)是白藜芦醇苯丙氨酸代谢合成的最后两个关键酶。运用悬挂PCR(overlap PCR)的方法将烟草4CL基因(Nt4CL)和虎杖STS基因(Pc PKS5)用3个中性氨基酸链连接,得到融合基因Nt4CL-Pc PKS5,将其插入原核表达载体中,构建p ET30a-Nt4CL-Pc PKS5重组质粒,表达Nt4CL-Pc PKS5融合蛋白。经Ni2+纯化和PD-10柱脱盐后,得到可溶性纯化蛋白。体外酶促反应结果表明该融合酶具有4CL和STS的双重活性,其催化产物为白藜芦醇。酶促反应最适条件为:p H 6.5,反应温度为45℃。研究结果获得了有效催化白藜芦醇生物合成的双功能融合酶,为进一步利用融合酶基因转化工程菌株实现白藜芦醇工业化生产奠定了基础。     

核基质结合区对转爬山虎芪合酶基因烟草中产生白藜芦醇的作用

采用核基质结合区(MARs)来提高转芪合酶基因(STS)烟草(Nicotiana tabacum L.)中白藜芦醇产物的含量。MARs是细胞中能与核基质特异紧密结合的DNA片段,体外结合实验表明克隆自酵母的MARs序列能特异地与烟草核基质结合。芪合酶是白藜芦醇生物合成中的关键酶,用RT-PCR方法从川鄂爬山虎(Parthenocissus henryana(Hemsl.) Diels et Gilg)中克隆了与葡萄芪合酶基因有较高同源性的芪合酶编码区,将其置于CaMV35SW强启动子下,分别构建两侧带有MARs及不含MARs序列的表达载体,通过农杆菌介导转化烟草。Northern blot及HPLC等分析表明STS基因已整合至烟草染色体中并正常转录,且表达的外源芪合酶在烟草中可催化其底物合成白藜芦醇产物。与对照相比,MARs的存在使转芪合酶基因烟草中白藜芦醇的含量平均提高了约一倍。MARs在转芪合酶基因植物中的应用也为获得抗病性更强、白藜芦醇含量更高、更保健的转基因果蔬的研究奠定了基础。     

虎杖苯亚甲基丙酮合酶PcPKS2的表达纯化和晶体生长

植物类型Ⅲ聚酮合酶超家族(PKSs),又称查尔酮合酶(Chalcone synthase,CHS)超家族,催化合成多种植物次生代谢产物的分子骨架。苯亚甲基丙酮合酶(Benzalacetone synthase,BAS)催化4-香豆酰辅酶A与丙二酰辅酶A通过一步脱羧缩合反应生成苯亚甲基丙酮,是一系列具有重要生物学活性苯丁烷类化合物及其衍生物的前体化合物。前期工作从虎杖中分离出苯亚甲基丙酮合酶BAS(PcPKS2)和1个具有CHS和BAS活性的双功能酶(PcPKS1)。两者与超家族其他成员序列经比较,在包括门卫氨基酸Phe215和Phe265在内的重要氨基酸序列存在一定差异。已有蛋白晶体学研究结果表明,PKSs家族不同成员的功能多样性来自于酶催化位点的非常微小的构象变化。为了能够从结构上比较PcPKS2和Pc PKS1双功能酶活性差异可能产生的机制,以确定其高效BAS活性的分子机理,研究利用了大肠杆菌原核表达系统过量表达了C-端融合有His6标签的重组蛋白,经纯化得到了高纯度蛋白。经过对其晶体生长条件进行摸索和优化,得到了能用于X-射线衍射的单晶,为其结构解析、催化机理研究、了解虎杖聚酮类化合物生物合成机制和该类酶在基因工程中的应用提供了基础。     

定点突变提高虎杖聚酮合酶的苯亚甲基丙酮合酶活性北大核心CSCD

虎杖(Polygonum cuspidatum)聚酮合酶(polyketide synthase 1,PcPKS1)同时具有查尔酮合酶(chalcone synthase,CHS)及苯亚甲基丙酮合酶(benzylidene acetone synthase,BAS)催化活性,能够催化生成聚酮类化合物柚皮素查尔酮和苯亚甲基丙酮,进而催化合成黄酮类或覆盆子酮等具有多种生物学活性的化合物。本研究通过分析虎杖PcPKS1与掌叶大黄(Rheum palmatum)BAS、拟南芥(Arabidopsis thaliana)CHS等家族成员的序列以及酶催化位点的构象,确定可能影响酶功能的3个氨基酸位点:Thr133、Ser134、Ser339。采用定点突变对PcPKS1进行分子修饰,成功获得2个突变体并进行相关体外酶促反应,高效液相色谱(high performance liquid chromatography,HPLC)产物分析结果表明,在pH 7.0和pH 9.0的体外酶促条件下,突变体T133LS134A和S339V维持BAS和CHS双功能活性,且BAS活性显著高于原PcPKS1。本研究为利用PcPKS1进行基因工程调节黄酮类和覆盆子酮化合物的生物合成提供理论依据。     

葡萄CHS和STS基因家族生物信息学鉴定和表达分析

查尔酮合成酶(CHS,chalcone synthase)是植物体类黄酮类化合物合成的第1个关键酶和限速酶,它能够催化丙二酰-Co A和对香豆酸-Co A合成柚皮素查尔酮。二苯乙烯合成酶(STS,stilbene synthase)是芪类化合物合成路径的关键酶,与查尔酮合成酶有共同的作用底物,二者具有很高的相似度。为更好地了解葡萄中CHS和STS基因的种类和数量,本研究采用生物信息学方法检索获得葡萄(Vitis vinifera L.)基因组数据库中的CHS和STS基因,通过分析其染色体定位、系统进化和保守基序,发现葡萄基因组可能含有33个STS基因,9个CHS基因,这些基因集中分布在6条葡萄染色体上,部分家族基因在染色体上形成基因簇。葡萄CHS和STS基因家族蛋白长度、基因结构和蛋白基序非常保守,具有很近的进化关系。葡萄芯片数据结果表明,葡萄CHS和STS基因在葡萄果实不同发育时期的果皮和果肉中均有表达,尤其葡萄CHS GroupsⅢ亚家族基因在葡萄果皮中大量表达。葡萄STS基因家族在果实中的表达量较低,部分探针在葡萄果实成熟期的果皮中表达量急剧增加。本研究结果可为葡萄CHS和STS基因在果实发育过程中的功能研究提供参考。     

异源表达CiRS基因拟南芥的黄酮代谢及抑菌能力研究

白藜芦醇合成酶(resveratrol synthase,RS)是查耳酮合酶基因家族的一个重要酶,在植物体内催化白藜芦醇的生成。白藜芦醇是植物产生的一种非黄酮多酚类代谢产物,是植物在受到生物和非生物胁迫时产生的植物抗毒素,已证实具有多种生理活性。从转录组数据库中筛选获得注释为CHS基因的CDS序列,以中间锦鸡儿cDNA为模板,克隆得到基因全长。序列分析、系统进化分析和转该基因拟南芥研究结果表明,该基因为RS基因,因此将其命名为CiRS(GenBank登录号MF678590)。qRT-PCR检测分析发现,中间锦鸡儿CiRS基因的表达受到干旱、NaCl、紫外线等胁迫诱导。异源表达CiRS基因抑制了拟南芥自身At CHS基因的表达。同时转CiRS基因拟南芥的抑菌活性强于野生型。这些结果均证实了中间锦鸡儿CiRS基因在转基因拟南芥中发挥了相应的功能。     

芪合酶基因的分子生物学研究

植物中存在芪类次生代谢产物(stilbenes)作为一种重要的植保素,不仅能够使植物体本身的抗逆性提高,在人类健康医疗领域也有很好的应用前景.由于其合成途径具有专一性,需要芪合酶(Stilbene synthase,STS)的存在,近年来芪合酶基因工程日益引起人们的研究和重视.介绍了芪合酶基因的结构功能及其诱导表达的调控机理,并对其转基因工程的研究进展进行了综述,以期为进一步开展芪类次生代谢物在作物品质改良及人类健康营养中的应用提供参考.     

引物悬挂延伸PCR扩增白藜芦醇合酶cDNA

为了得到葡萄白藜芦醇合酶(RS)基因的cDNA,实验以葡萄叶片为材料,利用引物悬挂延伸PCR法,以葡萄总DNA为模板,先分别扩增得到编码白藜芦醇合酶的第一外显子和第二外显子的序列,再以这两个序列为模板,进行第二轮的PCR,得到大小与RS基因大小相同的片段,经PCR产物测序证明,两个外显子已经准确无误地拼接,此片段就是RS基因。     

白藜芦醇合成酶基因在基因工程中的应用及功能研究进展

白藜芦醇合成酶(Resveratrol synthase,RS)是白藜芦醇(Resveratrol,Res)合成途径中的关键酶。以往研究报道,RS基因已在多种植物和微生物中进行了转化和表达,并在植物的代谢及调控等方面发挥生物学作用。文中主要围绕RS基因对植物的转化,及异源表达后植物体内代谢产物的变化,转RS基因对植物抗病原菌活性、抗自由基活性和生长发育的影响,以及利用RS基因在微生物中生产Res的相关进展进行了综述。并对RS基因在生物工程方面的应用前景进行展望。     

Two type III polyketide synthases from Polygonum cuspidatum: gene structure, evolutionary route and metabolites

In our recent work (Ma et al., in Planta 229(3):457–469, 2009a and 229(4):1077–1086, 2009b), two three-intron type III PKS genes, PcPKS1 and PcPKS2, were isolated from Polygonum cuspidatum Sieb. et Zucc. Phylogenetic and functional analyses revealed PcPKS1 is a three-intron chalcone synthase (CHS) gene, and PcPKS2 is found to be a three-intron benzalacetone synthase (BAS) gene. The regular CHS encoded by a single intron gene have not been isolated and characterized from P. cuspidatum. In this work a further CHS with one intron (PcPKS3) and a stilbene synthase (STS) gene with three-intron (PcPKS5) were isolated and characterized by functional and phylogenetic analyses. In comparison with PcPKS1, a bifunctional enzyme with both CHS and BAS activity, the enzymatic product of recombinant PcPKS3 was naringenin, bis-noryangonin (BNY) and 4-coumaroyltriacetic acid lactone (CTAL) occurred as side products. The PcPKS5 synthesized resveratrol and a trace amount of naringenin from p-coumaroyl-CoA. To our knowledge, PcPKS5 is the first reported three-intron STS gene in flowering plants. In this work, we speculated that this involved a possible evolutionary route of plant-specific type III PKS superfamily in P. cuspidatum.     

大肠杆菌中从头合成白藜芦醇途径的设计及优化

白藜芦醇是一种极具药用价值的植物源芪类化合物。为了在E. coli实现白藜芦醇的从头合成,构建了由酪氨酸解氨酶（TAL）,香豆酸-CoA合成酶（4CL）和白藜芦醇合成酶（STS）组成的非天然合成途径。经3天发酵后,白藜芦醇产量仅为2.67 mg/L,而其中间体香豆酸的积累达到了95.64 mg/L。为了进一步改善异源途径的效率,对4CL和STS模块采取融合表达、高拷贝表达及启动子工程改造的策略,最终使白藜芦醇产量提高到了9.6倍,达到了25.76 mg/L,同时香豆酸的积累减少到了20.38 mg/L。这些研究结果为更高效白藜芦醇从头合成工程菌的构建及最终实现白藜芦醇的微生物大规模生产奠定了基础。     

Expression pattern, genomic structure, and promoter analysis of the gene encoding stilbene synthase from Chinese wild Vitis pseudoreticulata

The gene encoding stilbene synthase (STS) plays a central role in many biochemical and physiological actions, and its metabolite resveratrol possesses broad-spectrum resistance to pathogens, as well as diverse pharmacological properties, notably an anticancer effect. Here, we report the expression analysis of the gene encoding STS and its promoter function from a powdery mildew (PM)-resistant Chinese wild Vitis pseudoreticulata, and compare it with two PM-susceptible cultivated grapevines, Vitis vinifera cvs. Carignane and Thompson Seedless. We show an unusual expression pattern of STS in V. pseudoreticulata, which differs markedly from that of the cultivated species. Sequence comparisons reveal that the genomic DNA sequences encoding STS in the three grapevines are highly conserved, but a novel residue mutation within the key motif of STS is solely present in V. pseudoreticulata. Moreover, the STS promoter in V. pseudoreticulata displays a significantly different structure from that found in the two V. vinifera. The three promoter-driven GUS differential expression patterns in transformed tobacco plants induced with Alternaria alternata, methyl jasmonate, and wounding indicated that the structurally different STS promoter of V. pseudoreticulata is responsible for its specific regulatory function. We also demonstrate that the expression of STS genes from their native promoters are functional in transformed tobacco and retain pathogen inducibility. Importantly, the genomic DNA-2 of V. pseudoreticulata under its native promoter shows good induction and the maximum level of resveratrol content. These findings further our understanding of the regulation of STS expression in a resistant grapevine and provide a new pathogen-inducible promoter system for the genetic improvement of plant disease resistance.     

Cross-reaction of chalcone synthase and stilbene synthase overexpressed in Escherichia coli.

Chalcone synthase (CHS) and stilbene synthase (STS) are related plant polyketide synthases belonging to the CHS superfamily. CHS and STS catalyze common condensation reactions of p-coumaroyl-CoA and three C(2)-units from malonyl-CoA but different cyclization reactions to produce naringenin chalcone and resveratrol, respectively. Using purified Pueraria lobata CHS and Arachis hypogaea STS overexpressed in Escherichia coli, bisnoryangonin (BNY, the derailed lactone after two condensations) and p-coumaroyltriacetic acid lactone (the derailed lactone after three condensations) were detected from the reaction products. More importantly, we found a cross-reaction between CHS and STS, i.e. resveratrol production by CHS (2.7-4.2% of naringenin) and naringenin production by STS (1.4-2.3% of resveratrol), possibly due to the conformational flexibility of their active sites.     

Pterostilbene production by microorganisms expressing resveratrol O-methyltransferase

Pterostilbene (3,5-dimethoxy-4′-hydroxyl-trans-stilbene)—a derivative of resveratrol—is a natural dietary compound and the primary antioxidant component in berries. Pterostilbene has significant advantages over resveratrol in bioavailability, half-life in the body, cellular uptake, oral absorption and metabolic stability. Here, we expressed the resveratrol O-methyltransferase (ROMT) gene (VvROMT) from grape (Vitis vinifera) in Escherichia coli and Saccharomyces cerevisiae and confirmed its specific ability to catalyze the production of pterostilbene from resveratrol. By co-expressing an additional two genes from the resveratrol biosynthetic pathway—4-coumarate CoA-ligase (4CL) and stilbene synthase (STS)—a large amount of pterostilbene was produced, with a trace amount of pinostilbene detected. To understand the molecular basis of the catalytic activity, four key amino acid residues were identified in a 3D-model of VvROMT and mutagenized and assayed for augmented catalytic activity. Our results demonstrate the potential utility of the engineered microorganisms for pterostilbene production and provide protein engineering targets that will hopefully lead to increased activity of the ROMT enzyme.