大豆异黄酮生物合成与调控的分子机制研究进展

大豆异黄酮,是由肉桂酰辅酶A产生的3-苯基衍生物,属植物次生代谢产物。它具有降血压、缓解更年期综合症等诸多功效,被称为"植物雌激素"。本文在阐述影响大豆子粒中异黄酮含量的QTL位点的基础上,重点综述了异黄酮生物合成途径中的多种酶和相关基因,以及调控该途径中相关酶系基因表达的转录因子和相关基因,并分析探讨了该领域一些亟待解决的问题和未来发展的方向,以期为深入研究大豆异黄酮合成的分子机制及高异黄酮大豆分子育种提供理论参考。     

合成大豆异黄酮的植物基因工程研究进展

异黄酮主要存在于豆科植物中,是一种具有广谱抗菌活性的黄酮类物质。结合本课题组的工作,综述以基因工程的方法在植物内合成异黄酮的研究历史与进展,重点深入分析和讨论异黄酮生物合成所受到的分子调控。     

植物芪类次生代谢物的功能、合成调控及基因工程研究进展

芪类(stilbenes)是松树、葡萄和花生等植物中发现的具有抗病及保健等多种功能的次生代谢物。芪合酶是芪类次生代谢物合成途径的关键酶。植物大多数芪类次生代谢物都具有诱导合成的特性，它们的诱导合成是芪合酶基因在各种诱发因子作用下被诱导转录和翻译的结果。利用已克隆的芪合酶基因，可在转基因植物中引入芪类合成支路以合成芪类次生产物，从而达到改变转基因植物抗性及保健品质的目的。     

浙贝母HMGR基因保守区序列的克隆及生物信息学分析

3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR)是甲羟戊酸途径中的第一个限速酶,在植物生物碱类物质代谢过程中发挥重要的作用。为探究HMGR酶在浙贝母甾体类生物碱合成代谢途径中的调控作用,以百合科浙贝母狭叶品种为材料,利用RT-PCR技术和Ta克隆技术首次从浙贝母中克隆出HMGR基因保守区序列,长度为390 bp。序列分析表明,浙贝母HMGR基因保守区序列与其他9种植物的HMGR基因有较高的相似性(84%~79%);蛋白质同源比对、特征区及系统进化树分析表明,浙贝母HMGR氨基酸片段与其他植物有较高的同源性(95%~88%),与球药隔重楼和海枣等单子叶植物的亲缘关系较近,据此,初步确定该基因为浙贝母HMGR基因,命名为Ft HMGR。Ft HMGR基因的克隆及分子鉴定为进一步解析该基因在浙贝母甾体类生物碱合成代谢途径中的分子调控作用奠定了基础,有助于对浙贝母活性物质的合成进行调控,从而提高产量。     

陆地棉法尼基焦磷酸合酶基因的克隆及表达分析

棉酚是棉花重要的次生代谢产物之一,具有抗虫及抗病害的作用。法尼基焦磷酸合酶是杜松烯合酶的上游酶,催化合成法尼基焦磷酸,该物质是棉酚等次生代谢物生物合成的前体。本文依据亚洲棉法尼基焦磷酸合酶基因序列,采用同源RT-PCR技术,克隆了陆地棉法尼基焦磷酸合酶基因,对其序列和编码产物结构进行了分析。荧光定量PCR分析表明,该基因在棉花植株叶片表达量最高,根部最低,在花、铃、蕾、茎中的表达水平接近。以上工作为棉花异戊二烯基途径的分析及相关次生代谢的途径工程研究奠定了一定的理论基础。     

生物表面活性剂生物合成的研究进展

生物表面活性剂是由微生物产生的具有高表面活性的生物分子,相对于化学合成的表面活性剂,生物表面活性剂对生态系统的毒性较低,且可生物降解。因此,其在工业生产上有潜在的广泛应用价值,人们关于其合成方面的研究逐年增多。但是,由于其是次生代谢产物,传统方法主要是进行高效菌株的筛选,以及研究不同发酵条件对其产量的影响。近年来,由于基因工程技术的广泛应用,分子生物学、遗传学和酶学等方面的方法被人们运用于生物表面活性剂合成过程的研究,人们了解了催化生物表面活性剂合成的各种关键酶、调控基因的操纵元结构、合成中底物变化的过程等,从而对其生物合成的过程有了更进一步了解。     

家种羌活主要化合物生物合成途径的转录组学研究

羌活是我国传统常用大宗药材，以根茎及根入药，前期研究发现其根茎中以羌活醇和异欧前胡素为代表的次生代谢产物含量显著高于根。为探究其生物合成途径及调控机制，本研究利用RNA-seq和转录组学分析手段，以根为对照，对比分析家种羌活根茎中基因的差异表达。结果表明，1 222个差异表达基因（DEGs）显著富集于苯丙烷生物合成、植物病原体相互作用、植物MAPK信号通路及植物激素信号转导途径等通路；苯丙烷生物合成通路中莽草酸O-羟基肉桂酰转移酶（HCT）、肉桂醇脱氢酶（CAD）和过氧化物还原酶6(PRDX6)等关键酶编码基因转录本在根茎中显著上调表达；AP2/ERF、WRKY、C2C2及NAC等转录因子家族的多个转录因子在根茎及根中显著差异表达，这些因子可能是影响羌活次生代谢产物生物合成的关键转录因子；基因的差异表达与羌活根茎及根中羌活醇和异欧前胡素含量差异相符。本研究结果可为进一步研究羌活药用部位有效成分积累机理及定向育种提供理论基础。     

用化肥开启植物次生代谢途径的原理和方法

1 植物生理学中关于胁迫作用的研究和应用 1.1 植物次生代谢途径是植物对胁迫的应激反 植物次生代谢途径是植物对胁迫的应激反应,它是在胁迫压力下进化而形成的--植物的次生代谢途径是植物用以适应不断深入的环境的结果,而这个能力必然在胁迫下再现.     

谷子硫解酶基因家族鉴定及表达模式分析

硫解酶(thiolase)是脂肪酸代谢的关键酶,在植物次生代谢合成、生长发育以及抵抗非生物胁迫中均发挥重要作用.为探究谷子硫解酶基因家族的基本特征,本研究利用生物信息学方法,对谷子硫解酶基因家族成员进行了鉴定,对蛋白理化性质及系统进化、基因结构、染色体位置、启动子、基因进化进行了分析;采用转录组测序进行了根、茎、叶、穗...     

大豆光能高效利用的分子调控机制研究进展

光合作用是影响作物产量的重要因素,长期以来国内外科学家通过高光效育种对大豆产量的提高起到了重大的促进作用。近年来,随着植物光合分子生物学研究和基因工程技术的发展,有关大豆光合作用分子机制和光能利用效率分子调控的研究取得了很多进展。本文总结了大豆光合作用相关的基因,综述了调控光能利用效率相关因子的研究进展,以期为大豆高光效分子设计育种提供理论指导。     

Phytochemicals for health, the role of pulses

Pulses are the seeds of legumes that are used for human consumption and include peas, beans, lentils, chickpeas, and fava beans. Pulses are an important source of macronutrients, containing almost twice the amount of protein compared to cereal grains. In addition to being a source of macronutrients and minerals, pulses also contain plant secondary metabolites that are increasingly being recognised for their potential benefits for human health. The best-studied legume is the soybean, traditionally regarded as an oilseed crop rather than a pulse. The potential health benefits of soy, particularly with respect to isoflavone content, have been the subject of much research and the focus of several reviews. By comparison, less is known about pulses. This review investigates the health potential of pulses, examining the bioactivity of pulse isoflavones, phytosterols, resistant starch, bioactive carbohydrates, alkaloids and saponins. The evidence for health properties is considered, as is the effect of processing and cooking on these potentially beneficial phytochemicals.     

Metabolism of biochanin A and formononetin by human liver microsomes in vitro

Biochanin A and formononetin are abundant in legumes. These proestrogenic isoflavones can be converted by 4'-O-demethylation to the more potent phytoestrogens genistein and daidzein. Incubation of biochanin A or formononetin with human liver microsomes resulted in 4'-O-demethylation and the production of additional metabolites. Three new hydroxylated formononetin derivatives, 6,7-dihydroxy-4'-methoxyisoflavone, 7,8-dihydroxy-4'-methoxyisoflavone, and 7,3'-dihydroxy-4'-methoxyisoflavone, were isolated and characterized. We surveyed the O-demethylase competence of cytochrome P450 isoforms found in human liver. Human cytochrome P450 isoforms 1A2, 2E1, 2C9*1, 2C19, and 2D6*1 catalyzed biochanin A consumption and genistein production. Human cytochrome P450 isoforms 1A2, 2C9*1, 2A6, 2D6*1, and 2C19 catalyzed formononetin consumption and daidzein production. These isoforms also generated other hydroxylated metabolites. Although O-demethylation of isoflavones has been attributed to metabolism by gut microflora, our study demonstrates that human hepatic microsomal enzymes can perform the same transformation and may play a key role in the conversion of 4'-O-methylated isoflavones to more potent phytoestrogens.     

Characterization of isoflavones and their conjugates in female rat urine using LC/MS/MS

Isoflavone phytoestrogens found in soybeans are the most widely studied phytochemicals in human diets and soy infant formulas. The health benefits of the isoflavones daidzein and genistein have been reported, and concerns about potential adverse effects have also been raised. However, the results of direct analysis of isoflavones and their metabolites in biological fluids after consumption of soy-containing diets are scarce. This study describes an LC/MS/MS method for the analysis of isoflavones and their metabolites in the urine of female rats fed diets made with soy protein isolate. Five isoflavones (daidzein, genistein, glycitein, dihydrodaidzein, and O-desmethylangolensin) were identified by comparison with authentic standards. Seventeen conjugates of isoflavones were characterized in the urine, the most unusual being genistein 5-glucuronide and four glucuronide conjugates of reductive metabolites of daidzein. The application of LC/MS/MS to analyze isoflavone metabolites is simple and sensitive, and appears to be an excellent method for determining the bioavailability and metabolism of food phytochemistry.     

Utilization of the genetic resources of wild species to create a nontransgenic high flavonoid tomato

Flavonoids represent a large and important group of plant natural products that are ubiquitous in the plant kingdom. Epidemiological studies have shown the health benefits of a diet high in flavonoids. However, the dietary intake of flavonoids in most western populations is limited, creating a need to find alternative food sources for these polyphenolic secondary metabolites. The domestication of many of our cultivated food crops has resulted in alterations in the biosynthetic pathways of many essential micronutrients and vitamins through inadvertent counterselection against nutritional traits in favor of agronomic ones. Flavonoids are nearly absent from fruits of cultivated tomato (Lycopersicon esculentum Mill.), a major vegetable in human diets. Previous attempts to restore the flavonoid pathway in tomato fruits have been limited to transgenic strategies, suggesting that the problem was intractable through traditional methods. Here, we describe for the first time a nontransgenic metabolic engineering approach to developing a high flavonoid tomato using a wild tomato species (Lycopersicon pennelliiv. puberulum) and demonstrate the opportunities for restoring functional pathways using the genetic resources of wild species, resulting in production of healthier foods.     

Studies of the in vitro intestinal metabolism of isoflavones aid in the identification of their urinary metabolites

Soy isoflavones have recently gained considerable interest due to their possible health benefits. However, detailed studies on the metabolism of isoflavones are lacking. The aims of the investigation presented here were (1) to study the in vitro intestinal metabolism of isoflavones and their hydroxylated analogues 3'-OH-daidzein, 6-OH-daidzein, 8-OH-daidzein, and 3'-OH-genistein and (2) to characterize the structures of some earlier identified urinary metabolites of soy isoflavones, for which no authentic reference compounds have been available. Isoflavone standards (1-2 mg) were fermented with human fecal flora (16.7%) for 24 h. Metabolites formed during the fermentation were tentatively identified by interpretation of the mass spectra of trimethylsilylated compounds obtained by GC-MS. Compounds having hydroxyl groups at 5-position (i.e., genistein and 3'-OH-genistein) were completely converted to metabolites that could not be detected by the methods used in this study. The metabolism of daidzein and its hydroxylated analogues, 3'-OH-daidzein, 6-OH-daidzein, and 8-OH-daidzein, occurred to a much lesser extent. Minor amounts of reduced metabolites (i.e., isoflavanones and alpha-methyldeoxybenzoins) of these compounds were tentatively identified in fermentation extracts. The retention times and the mass spectra of reduced isoflavone metabolites, obtained from in vitro fermentations of pure compounds, were utilized to identify unknown urinary metabolites of soy isoflavones. Four novel isoflavone metabolites were identified in human urine collected after soy supplementation: 3' '-OH-O-desmethylangolensin, 3',4',7-trihydroxyisoflavanone, 4',7,8-trihydroxyisoflavanone, and 4',6,7-trihydroxyisoflavanone.     

Isoflavonoid composition of a callus culture of the relict tree Maackia amurensis Rupr. et Maxim

Isoflavonoids, an interesting and restricted group of secondary metabolites of legumes, exhibit estrogenic, antiangiogenic, and anticancer activities and are now popular as dietary supplements. Plant cell cultures that possess an increased ability to synthesize these metabolites were examined. During the investigation, cell cultures of the Far Eastern relict tree Maackia amurensis (Leguminosae) were established. A selection of seed-derived cell aggregates yielded the callus line designated A-18. This culture produces 20 isoflavonoids, namely, the isoflavones genistein, daidzein, formononetin, calycosin, derrone, and pseudobaptigenin and their glycosylated conjugates genistin, 6'-O-malonylgenistin, ononin, 6'-O-malonylononin, daidzin, 3'-methoxydaidzin, 4'-O-beta-D-glucopyranosyldaidzin, 4'-O-beta-D-glucopyranosylgenistin, and 7-O-beta-D-glucopyranosylcalycosin; the pterocarpans maackiain and medicarpin and their glycosylated conjugates 6'-O-malonyl-3-O-beta-D-glucopyranosylmaackiain and 6'-O-malonyl-3-O-beta-D-glucopyranosylmedicarpin; and the new pterocarpan glucoside 6'-O-malonyl-3-O-beta-D-glucopyranosyl-6,6a-dehydromaackiain. These isoflavonoids, possessing a hepatoprotective activity, were stably produced by the A-18 calli for prolonged periods of observation.     

Identification of urinary metabolites of the red clover isoflavones formononetin and biochanin A in human subjects

Dietary supplements manufactured from red clover are widely marketed to provide the beneficial health effects of isoflavones without changing the original diet. In this study the metabolism of formononetin and biochanin A, the principal isoflavones of red clover, was studied in human subjects. Seven women ingested four red clover dietary supplements, and the metabolites of the isoflavones were identified in their urine samples. The structures of trimethylsilyl derivatives of the metabolites were established by GC-MS. New reduced metabolites of formononetin (dihydroformononetin and angolensin) and biochanin A (dihydrobiochanin A and 6'-hydroxyangolensin) were identified in urine samples using authentic reference compounds. Possible metabolic pathways are presented for the red clover isoflavones formononetin and biochanin A.     

Absorption and plasma disposition of genistin differ from those of genistein in healthy women

The chemical forms in which isoflavones appear in food or supplements seem to play an important role in their absorption efficiency. However, the influence of the chemical form of isoflavones on their plasma disposition has never been reported, although the metabolites of isoflavones circulating in the blood may have biological activity themselves. The purpose of the study was to investigate the pharmacokinetic profiles of genistein (GEN) and its phase II metabolites in the plasma and urine of healthy young women after multiple doses of pure aglycone and glucoside forms of GEN. Genistein-7-glucuronide (G-7-G), 4'-glucuronide (G-4'-G), 7-sulfate (G-7-S), 4'-sulfate (G-4'-S), 4',7-diglucuronide (G-4',7-diG), and 7-glucuronide-4'-sulfate (G-7-G-4'-S) besides unconjugated GEN were observed in human plasma after ingestion of GEN and its glucoside. Among these metabolites, G-4',7-diG and G-7-G-4'-S were the major ones, comprising both about 30% of the total amount of GEN in plasma. Compared with the aglycone, the amount of total GEN in vivo and those of G-4',7-diG and G-7-G-4'-S were increased after the glucoside intake. No difference was observed in urinary excretion between the aglycone and the glucoside. Overall, the absorption and plasma disposition of GEN were affected by the glucoside form.     

Antioxidant activity of isoflavones and their major metabolites using different in vitro assays

Isoflavone phytoestrogens found mainly in soybeans and clover are widely studied phytochemicals. Genistein and daidzein, the major isoflavones found in soy, have received the most attention. However, they undergo extensive metabolism in the intestine and the liver, which might affect their biological properties, e.g. their antioxidant capacities. Furthermore, the biological activities of other naturally occurring isoflavones, for instance, glycitein from soy or biochanin A from red clover, have not yet been studied in detail. The aim of this study was to investigate the antioxidant activities of six naturally occurring isoflavones and their corresponding oxidative and bacterial metabolites. The oxygen radical absorbance capacity assay as well as the in vitro oxidation of low density lipoproteins with the conjugated diene and the thiobarbituric acid reacting substances formation as end points were used. The oxidative metabolites of genistein and daidzein as well as equol exhibited the highest antioxidant activities in all three assays. With few exceptions, they were more effective than the positive controls quercetin and ascorbic acid. Formononetin, the 4'-O-methyl ether of daidzein, showed the lowest antioxidant property. Because the antioxidant efficacy of isoflavones as effective antioxidants is evident at concentrations well within the range found in the plasma of subjects consuming soy products, this biological activity could be of physiological relevance.