硫代葡萄糖苷及其降解产物异硫代氰酸盐

硫代葡萄糖苷是一种含硫的次级代谢产物,广泛分布于十字花科植物中。不同的栽培种、不同的生理阶段、不同的组织部位以及不同的栽培条件,都会使植物中含有的硫代葡萄糖苷的含量和成分有所变化。当硫代葡萄糖苷经葡萄糖硫苷酶作用时会发生降解,生成异硫代氰酸盐等产物。采后的一系列处理会影响植物中硫代葡萄糖苷的含量。硫代葡萄糖苷的降解产物异硫代氰酸盐作为一种化学预防剂,能抑制阶段Ⅰ酶（phase Ⅰ enzyme）,诱导阶段Ⅱ酶（phaseⅡenzyme）,从而防止癌症的发生。目前对硫代葡萄糖苷的鉴定方法主要是高效液相色谱法,气相色谱法等。     

菜籽粕脱毒液中硫代葡萄糖苷提取工艺的研究

本文用两步萃取法从菜籽粕脱毒液中提取硫代葡萄糖苷,以3,5-二硝基水杨酸比色法测定硫代葡萄糖苷含量,采用单因素试验法,分别考察可能影响硫代葡萄糖苷提取效果的各种因素,确定了最佳工艺条件：第一步萃取剂为异辛烷,母液与异辛烷体积比为2：1,搅拌时间为30min,异辛烷萃取次数为2次,异辛烷单次萃取率达71．2％。硫代葡萄糖苷收率为83．7％。     

黑芥子酶研究进展

黑芥子酶又称黑芥子硫苷酸酶,广泛存在于自然界。它能催化硫代葡萄糖苷水解成为葡萄糖和不稳定的中间物配基,此配基易重排形成异硫氰化合物,硫氰化合物,腈,口恶唑烷硫酮等有毒物质。当植物受到昆虫,哺乳动物或病源伤害时,黑芥子酶与硫代葡萄糖苷混合,释放有毒的水解产物。因此认为黑芥子酶-硫代葡萄糖苷系统是植物重要的防御系统。另一方面,黑芥子酶催化硫代葡萄糖苷所生成的有毒物质,在一定程度上也影响了十字花科油料及蔬菜作物的品质,因此,引起了对黑芥子酶及硫代葡萄糖苷研究的重视。本文仅对黑芥子酶的研究概况作一综述。1　黑芥子酶的…     

黑芥子酶研究进展

综述了黑芥子酶的研究进展,包括黑芥子酶催化硫代葡萄糖苷的水解产物对动植物和人体的作用;黑芥子酶的性质、提纯技术、活性测定、同工酶的基因家族;产黑芥子酶的植物、动物和微生物等。部分硫代葡萄糖苷可经黑芥子酶水解成有抗癌作用的异硫代氰酸盐。可通过测定葡萄糖的生成量或底物硫代葡萄糖苷浓度的变化来确定黑芥子酶的活性。十字花科等植物,以十字花科植物为食的拟步行虫、黄条跳甲、甘蓝蚜虫、小菜蛾、粉纹夜蛾等昆虫,啤酒酵母、交链孢霉属、茎点霉等真菌,以及多型拟杆菌等肠道细菌都有黑芥子酶。     

西兰花中β-硫代葡萄糖苷总量的测定

通过内源和外源芥子酶酶解硫代葡萄糖苷产生的葡萄糖的测定,间接测出西兰花中β-硫代葡萄糖苷总量,并对测定的影响因素进行了研究,与吡啶滴定法对比实验表明,该法准确可靠,操作简便。     

植物芥子酶研究进展

芥子酶防御系统为白花菜目植物特有．由芥子酶及其底物硫代葡萄糖苷组成．芥子酶和硫代葡糖苷分别储藏在不同的细胞中,在受到病虫侵袭时,底物和酶相遇,硫代葡糖苷被降为有毒化合物,起防御作用．对植物芥子酶防御系统研究进展进行了综述,包括基因家族的结构、基因的表达调控、芥子酶的细胞定位、植物以外其它生物的芥子酶、硫代葡糖苷／芥子酶系统起源进化以及其可能功能等．     

硫代葡萄糖苷合成核心途径与植物生长素微调

硫代葡萄糖苷是刺山柑Capparis spinosa(Capers)和十字花科(Brassicaceae)植物中一类特有和重要的次生代谢产物.在植物体内,硫代葡萄糖苷的合成包括前体氨基酸侧链延伸,核心结构的形成和次级侧链修饰3个步骤.利用核心结构合成途径上有关催化酶的基因突变体证明,后醛肟反应途径上任何催化酶基因的突变,都将导致吲哚乙醛肟结构相似的吲哚乙酸(IAA)含量变化,硫苷核心途径对生长素的动态平衡起着微调(fine-tune)作用.     

辣木种子主要成分分离制备及功能应用研究进展

辣木是亚洲和非洲广泛种植的一种药食同源植物。辣木生长快、种子产量高,每公顷辣木可产种子24 t。辣木种子含油19%～47%,蛋白质10%～52%,硫代葡萄糖苷及其水解产物异硫氰酸酯6.2%～8.6%。辣木油具有抗氧化、抗菌和抗炎作用,其不饱和脂肪酸含量高达71.7%～83.1%,是优质的食用油来源。辣木蛋白具有抗菌作用,是良好的食用和饲料用蛋白源。辣木种子所含硫代葡萄糖苷及其水解产物异硫氰酸酯有抗炎、抗肿瘤和降血糖等作用。辣木种子具有较高的开发利用价值。本文综述辣木种子上述四种主要成分分离制备和功能应用的研究进展,为辣木种子综合开发利用提供参考。     

野生大叶碎米荠中的异硫氰酸盐成分分析

大叶碎米荠为十字花科碎米荠属多年生草本植物,作为药食同源蔬菜,具有通尿利便、止痛及治败血病等药效。异硫氰酸盐是存在于十字花科植物中的一种具有防癌抗癌作用的有机硫化合物。本研究用二氯甲烷从大叶碎米荠水解液中提取出硫代葡萄糖苷水解产物,经气相-质谱联用分析其组分,鉴定出2种异硫氰酸盐化合物,分别为异硫氰酸烯丙酯和异硫氰酸甲硫基烯丙酯,两者的含量分别为2.32 mg/kg、0.50 mg/kg。该研究为大叶碎米荠作为野菜资源开发利用提供了科学依据。     

bFGF反义寡核苷酸增强鼠黑色素瘤B16细胞化疗药物敏感性研究

目的:研究bFGF反义硫代寡核苷酸增强肿瘤细胞对化疗药物敏感性作用。方法:设计、合成bFGF寡核苷酸,用聚乙烯亚胺(polyemyleneimine,PEI)介导bFGF反义硫代寡核苷酸转染入黑色素瘤B16细胞,MTT法检测bFGF反义硫代寡核苷酸及其与化疗药物联合处理后的细胞增殖率;半定量RT-PCR测定bFGF反义硫代寡核苷酸转染后细胞中bFGF mRNA水平;流式细胞仪分析bFGF反义硫代寡核苷酸诱导的细胞凋亡。结果:bFGF反义硫代寡核苷酸对B16细胞增殖的抑制率为64.8%,且呈剂量依赖效应。B16细胞中bFGF mRNA被bFGF反义硫代寡核苷酸显著降低,为对照细胞的57.9%,且bFGF反义硫代寡核苷酸诱导B16细胞凋亡,凋亡率为41.8%。bFGF反义硫代寡核苷酸转染能显著增强B16细胞对阿霉素、5-氟脲嘧啶及顺铂的敏感性,非特异性硫代寡核苷酸不影响阿霉素、5-氟脲嘧啶及顺铂抑制B16细胞增殖。结论:bFGF反义硫代寡核苷酸显著增强B16细胞的化疗敏感性,表明其可协同化疗药物用于治疗肿瘤。     

植物激素与芥子油苷在生物合成上的相互作用

植物激素在植物的生长发育中起着关键性作用,芥子油苷是一类重要的次生代谢物质。植物激素与芥子油苷之间存在复杂的相互作用。生长素与吲哚类芥子油苷在生物合成上存在着相互作用。植物防卫信号分子与芥子油苷之间也存在相互作用,茉莉酸强烈诱导吲哚类芥子油苷生物合成相关基因CYP7982和CYP7983的表达,从而诱导吲哚-3-甲基芥子油苷和N-甲氧吲哚-3-甲基芥子油苷等吲哚类芥子油苷的生成,水杨酸和乙烯则能轻度诱导4-甲氧吲哚-3-甲基芥子油苷的生成。植物防卫信号转导途径相互作用以精细调节不同种类吲哚类芥子油苷的生成。     

Cytochromes P450 in the biosynthesis of glucosinolates and indole alkaloids

Characteristic of cruciferous plants is the synthesis of nitrogen- and sulfur-rich compounds, such as glucosinolates and indole alkaloids. The intact glucosinolates have limited biological activity, but give rise to an array of bio-active breakdown products when hydrolysed by endogenous β-thioglucosidases (myrosinases) upon tissue disruption. Both glucosinolates and indole alkaloids constitute an important part of the defence of plants against herbivores and pathogens, with the difference that a basal level of glucosinolates is ever-present in the plant whereas indole alkaloids are true phytoalexins that are de novo synthesised upon pathogen attack. With the completion of the genome sequence of the model plant, Arabidopsis thaliana, which is a crucifer, many genes involved in the biosynthesis of glucosinolates and indole alkaloids have been identified and cytochromes P450 are key players in these pathways. In the present review, we will focus on the cytochromes P450 in the biosynthesis of both groups of compounds. Their functional roles and regulation will be discussed.     

CYP79F1 and CYP79F2 have distinct functions in the biosynthesis of aliphatic glucosinolates in Arabidopsis

Cytochromes P450 of the CYP79 family catalyze the conversion of amino acids to oximes in the biosynthesis of glucosinolates, a group of natural plant products known to be involved in plant defense and as a source of flavor compounds, cancer-preventing agents and bioherbicides. We report a detailed biochemical analysis of the substrate specificity and kinetics of CYP79F1 and CYP79F2, two cytochromes P450 involved in the biosynthesis of aliphatic glucosinolates in Arabidopsis thaliana. Using recombinant CYP79F1 and CYP79F2 expressed in Escherichia coli and Saccharomyces cerevisiae, respectively, we show that CYP79F1 metabolizes mono- to hexahomomethionine, resulting in both short- and long-chain aliphatic glucosinolates. In contrast, CYP79F2 exclusively metabolizes long-chain elongated penta- and hexahomomethionines. CYP79F1 and CYP79F2 are spatially and developmentally regulated, with different gene expression patterns. CYP79F2 is highly expressed in hypocotyl and roots, whereas CYP79F1 is strongly expressed in cotyledons, rosette leaves, stems, and siliques. A transposon-tagged CYP79F1 knockout mutant completely lacks short-chain aliphatic glucosinolates, but has an increased level of long-chain aliphatic glucosinolates, especially in leaves and seeds. The level of long-chain aliphatic glucosinolates in a transposon-tagged CYP79F2 knockout mutant is substantially reduced, whereas the level of short-chain aliphatic glucosinolates is not affected. Biochemical characterization of CYP79F1 and CYP79F2, and gene expression analysis, combined with glucosinolate profiling of knockout mutants demonstrate the functional role of these enzymes. This provides valuable insights into the metabolic network leading to the biosynthesis of aliphatic glucosinolates, and into metabolic engineering of altered aliphatic glucosinolate profiles to improve nutritional value and pest resistance.     

芥子油苷在植物-生物环境关系中的作用

芥子油苷是一类含氮、含硫的植物次生代谢物质,主要分布于白花菜目的十字花科植物。芥子油苷及其降解产物具有多种生化活性,近年来人们更多地关注芥子油苷代谢与其它物质代谢途径的相互联系以及与植物生存环境的相互作用。介绍了芥子油苷及其分布、芥子油苷-黑芥子酶系统以及由芥子油苷介导的植物对昆虫、病原体的防御作用和植物与植物之间的化感作用的研究概况。     

Localization of the glucosinolate biosynthetic enzymes reveals distinct spatial patterns for the biosynthesis of indole and aliphatic glucosinolates

Glucosinolates constitute the primary defense metabolites in Arabidopsis thaliana (Arabidopsis). Indole and aliphatic glucosinolates, biosynthesized from tryptophan and methionine, respectively, are known to serve distinct biological functions. Although all genes in the biosynthetic pathways are identified, and it is known where glucosinolates are stored, it has remained elusive where glucosinolates are produced at the cellular and tissue level. To understand how the spatial organization of the different glucosinolate biosynthetic pathways contributes to their distinct biological functions, we investigated the localization of enzymes of the pathways under constitutive conditions and, for indole glucosinolates, also under induced conditions, by analyzing the spatial distribution of several fluorophore‐tagged enzymes at the whole plant and the cellular level. We show that key steps in the biosynthesis of the different types of glucosinolates are localized in distinct cells in separate as well as overlapping vascular tissues. The presence of glucosinolate biosynthetic enzymes in parenchyma cells of the vasculature may assign new defense‐related functions to these cell types. The knowledge gained in this study is an important prerequisite for understanding the orchestration of chemical defenses from site of synthesis to site of storage and potential (re)mobilization upon attack.     

Metabolic engineering of valine- and isoleucine-derived glucosinolates in Arabidopsis expressing CYP79D2 from Cassava

Glucosinolates are amino acid-derived natural products that, upon hydrolysis, typically release isothiocyanates with a wide range of biological activities. Glucosinolates play a role in plant defense as attractants and deterrents against herbivores and pathogens. A key step in glucosinolate biosynthesis is the conversion of amino acids to the corresponding aldoximes, which is catalyzed by cytochromes P450 belonging to the CYP79 family. Expression of CYP79D2 from cassava (Manihot esculenta Crantz.) in Arabidopsis resulted in the production of valine (Val)- and isoleucine-derived glucosinolates not normally found in this ecotype. The transgenic lines showed no morphological phenotype, and the level of endogenous glucosinolates was not affected. The novel glucosinolates were shown to constitute up to 35% of the total glucosinolate content in mature rosette leaves and up to 48% in old leaves. Furthermore, at increased concentrations of these glucosinolates, the proportion of Val-derived glucosinolates decreased. As the isothiocyanates produced from the Val- and isoleucine-derived glucosinolates are volatile, metabolically engineered plants producing these glucosinolates have acquired novel properties with great potential for improvement of resistance to herbivorous insects and for biofumigation.     

Characterization of methylsulfinylalkyl glucosinolate specific polyclonal antibodies

Antibodies towards small molecules, like plant specialized metabolites, are valuable tools for developing quantitative and qualitative analytical techniques. Glucosinolates are the specialized metabolites characteristic of the Brassicales order. Here we describe the characterization of polyclonal rabbit antibodies raised against the 4-methylsulfinylbutyl glucosinolate, glucoraphanin that is one of the major glucosinolates in the model plant Arabidopsis thaliana (hereafter Arabidopsis). Analysis of the cross-reactivity of the antibodies against a number of glucosinolates demonstrated that it was highly selective for methionine-derived aliphatic glucosinolates with a methyl-sulfinyl group in the side chain. Use of crude plant extracts from Arabidopsis mutants with different glucosinolate profiles showed that the antibodies recognized aliphatic glucosinolates in a plant extract and did not cross-react with other metabolites. These methylsulfinylalkyl glucosinolate specific antibodies have prospective use in multiple applications such as ELISA, co-immunoprecipitation and immunolocalization of glucosinolates.     

Cytochrome p450 CYP79F1 from arabidopsis catalyzes the conversion of dihomomethionine and trihomomethionine to the corresponding aldoximes in the biosynthesis of aliphatic glucosinolates

Glucosinolates are natural plant products that have received rising attention due to their role in interactions between pests and crop plants and as chemical protectors against cancer. Glucosinolates are derived from amino acids and have aldoximes as intermediates. We report that cytochrome P450 CYP79F1 catalyzes aldoxime formation in the biosynthesis of aliphatic glucosinolates in Arabidopsis thaliana. Using recombinant CYP79F1 functionally expressed in Escherichia coli, we show that both dihomomethionine and trihomomethionine are metabolized by CYP79F1 resulting in the formation of 5-methylthiopentanaldoxime and 6-methylthiohexanaldoxime, respectively. 5-methylthiopentanaldoxime is the precursor of the major glucosinolates in leaves of A. thaliana, i.e. 4-methylthiobutylglucosinolate and 4-methylsulfinylbutylglucosinolate, and a variety of other glucosinolates in Brassica sp. Transgenic A. thaliana with cosuppression of CYP79F1 have a reduced content of aliphatic glucosinolates and a highly increased level of dihomomethionine and trihomomethionine. The transgenic plants have a morphological phenotype showing loss of apical dominance and formation of multiple axillary shoots. Our data provide the first evidence that a cytochrome P450 catalyzes the N-hydroxylation of chain-elongated methionine homologues to the corresponding aldoximes in the biosynthesis of aliphatic glucosinolates.     

Phylloplane location of glucosinolates in Barbarea spp. (Brassicaceae) and misleading assessment of host suitability by a specialist herbivore

Glucosinolates are plant secondary metabolites used in host plant recognition by insects specialized on Brassicaceae, such as the diamondback moth (DBM), Plutella xylostella. Their perception as oviposition cues by females would seem to require their occurrence on the leaf surface, yet previous studies have reached opposite conclusions about whether glucosinolates are actually present on the surface of crucifer leaves. DBM oviposits extensively on Barbarea vulgaris, despite its larvae not being able to survive on this plant because of its content of feeding-deterrent saponins. Glucosinolates and saponins in plant tissue and mechanically removed surface waxes from leaves of Barbarea spp. were analyzed with high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). Surface waxes from leaves of Barbarea spp. contained glucosinolates, but not feeding-deterrent saponins. Our research is the first to show that glucosinolates are present on the leaf surface of Barbarea spp., but not in other crucifers investigated, resolving some conflicting results from previous studies. Our research is also the first to quantify glucosinolates on the leaf surface of a crucifer, and to show that the concentrations of glucosinolates found on the leaf surface of Barbarea spp. are sufficient to be perceived by ovipositing DBM.     

The effect of modifying the glucosinolate content of leaves of oilseed rape (Brassica napus ssp. oleifera) on its interaction with specialist and generalist pests

Twenty eight Brassica napus lines were developed which had contrasting leaf glucosinolate profiles to those found in commercial oilseed rape cultivars. The lines varied both in the total amount of aliphatic glucosinolates and in the ratio of different side chain structures. The lines were used in field experiments to assess the manner by which glucosinolates mediate the interactions between Brasssica and specialist pests (Psylliodes chrysocephala and Pieris rapae) and generalist pests (pigeons and slugs). Increases in the level of glucosinolates resulted in greater damage by adult flea beetles (P. chrysocephala) and a greater incidence of Pieris rapae larvae, but reduced the extent of grazing by pigeons and slugs. Decreasing the side chain length of aliphatic glucosinolates and reducing the extent of hydroxylation of butenyl glucosinolates increased the extent of adult flea beetle feeding. The implications of modifying the glucosinolate content of the leaves of oilseed rape and the role of these secondary metabolites in plant/herbivore interactions are discussed.