摘要
黄酮类化合物系色原烷或色原酮的衍生物，其基本骨架具有C_6—C_3—C_6的特点，即指两个苯环(A与B环)通过中央三碳链相互连接而成的一系列化合物。它们的种类很多，包括黄酮、异黄酮、黄酮醇、查尔酮等。黄酮类化合物是一类在高等植物中大量存在的重要的次生代谢产物，它们有很多重要的功能，例如，参与花色的形成、UV防护、抵抗病原体、植物与微生物之间相互作用的信号分子、影响花粉生育率以及调节植物生长。目前研究表明，它们与人类的健康有着密切的关系。黄酮类化合物是药用植物中主要活性成分之一，具有消除氧自由基、抗氧化、抗炎、抗过敏、抗癌、雌激素样作用、保护心脑血管系统等多种药理作用。
查尔酮合成酶(chalcone synthase，CHS)是所有黄酮类化合物合成的关键酶，它介导了此类化合物合成的第一步。它催化3分子的丙二酰辅酶A(malonyl-CoA)与1分子对羟基苯丙烯酰辅酶A(4-coumaroyl-CoA)缩合成柚皮素查尔酮(naringenin-chalcone)，以此为支架其他黄酮类化合物得以衍生出来。目前，包括单子叶和双子叶植物的很多物种中，有的物种查尔酮合成酶已经得到纯化且基因组、cDNA克隆得到分离，有的或者得到了纯化酶，或者分离得到了基因组、cDNA克隆。在很多植物中，CHS的编码基因组成了一个多基因家族，例如，矮牵牛、豌豆、大豆等。不同物种中查尔酮合成酶氨基酸水平的同源性很高，79～91％。关于查尔酮合成酶结构的分析，不仅可以为研究植物中聚酮化合物合成的反应机制提供大量有用的信息，也为通过基因工程手段生成新的酶，并利用这些酶合成新的物质提供了可能性。查尔酮合成酶活性的积累与黄酮类化合物的积累是紧密相关的，因此对查尔酮合成酶的分子生物学研究可以为在农作物中提高黄酮类化合物的合成，增强农作物的抗病能力，同时产生对人类健康有益的食品奠定基础。
本研究利用RT-PCR及RACE方法，从黄蜀葵花中(Abelmoschus manihot(L.)Medicus)克隆得到一个新的含有1309个核苷酸的CHS基因，其最大的开放阅读框(ORF)有1173 bp，推测其蛋白由390个氨基酸组成。我们将该基因命名为AmCHS。通过在Gene Bank中BLAST及生物学软件中进行核苷酸和蛋白的同源序列比较，我们推测所得的基因为黄蜀葵CHS全长cDNA序列。构建了卡那霉素抗性的植物表达载体pC2TCHS，用农杆菌介导法将CHS基因转入了烟草NC89中，获得15株卡那霉素抗性的植株。检测其中的4株，PCR结果表明其中2株为阳性植株，说明黄蜀葵查尔酮合成酶基因已经整合到烟草基因组中。
Flavonoids, derivatives of chromone or chromane, are built upon a C6-C3-C6 flavone skeleton in which the three-carbon bridge between the phenyl groups is commonly cyclized with oxygen. There are diverse group of plants containing flavone, isoflavone, flavonol, chalcone, ect. Flavonoids form an important class of secondary metabolites which are abundant in all higher plants. They play important roles in flower pigmentation, protection against UV light, defence against phytopathogens, signal transduction between plants and microorgnism interactions, pollen fertilization and growing regulation. Flavonoids are the mostly active components in the officinal plants, and exhibit a wide range of human health-promoting activities that are currently the focus of intense study. Flavonoids have been found to own potent antioxidant and free radical scavenging activities in vitro. There is growing evidence from human consumption studies supporting the protective roles of flavonoids in cardiovascular diseases and cancer. Ma
ny flavonoids have been found to possess antiphlogistic, estrogenic or anti-allergenic properties.
Chalcone synthase (CHS) is the first committed enzyme in the flavonoid biosynthesis catalyzing the condensation of 3 moleculars of malonyl-CoA and one molecular of hydroxycinnamoyl-CoA ester to form a naringenin-chalcone intermediate. Further isomerization and substitution of this central intermediate leads to the synthesis of other flavonoids. CHSs has been isolated from different plant species, including both monocots and dicots, some of their genomic DNAs and/or cDNAs have also been isolated. CHS is encoded by a multigene family in many plants, such as Petunia, Pea, Soybean, and so on. The amino acid identity among CHSs from different species is high, ranging from 79% to 91 %. The three-dimensional structure of CHS elucidates the chemical basis of plant polyketide biosynthesis and provides a framework for engineering CHS-like enzymes to produce new products. Enhancing flavonoid biosynthesis in chosen crops may provide new raw materials that have the potential to be used in foods designed for specific bene
fits to human health.
In this study, a novel cDNA encoding chalcone synthase was cloned from the flower of Abelmoschus manihot (L.) Medicus by RT-PCR and RACE. The open read frame of the gene has 1173bp and encodes a polypeptide of 390 amino acids. We named this gene AmCHS. We presume this new cDNA is the CHS full-length cDNA by BLAST and alignment of nucleotide and amino acid. A plant constitutive expression vector under the control of CaMV35S promoter was constructed by inserting the AmCHS cDNA into pCAMBIA2301-pT Q 4A. The AmCHS gene was introduced to tobacco by Agrobacterium-mediated transfer method, and 15 individual kanamycin resistant plants have been obtained. Results of PCR analysis showed that the foreign AmCHS gene has been integrated into the tobacco genome in two transformants.
引文
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