高通量测序解析大青叶有效成分的生物合成途径

中药大青叶是菘蓝的干燥叶片,主要活性物质为吲哚类化合物,包括靛蓝、靛玉红、色胺酮等,以上吲哚类化合物的生物合成起始于色氨酸途径,目前在植物中的合成机制还未完全阐明.本研究以成熟菘蓝叶片为研究对象,对茉莉酸甲酯诱导的叶片进行了转录组分析,对已知途径进行了转录组注释,并对未解析途径进行了预测分析.分别获得了色氨酸代谢途径中色氨酸、吲哚乙酸、吲哚苷和萜类吲哚生物碱合成几个代谢支路中16个催化步骤的38个编码基因.通过共表达网络分析,预测转录因子bHLH125可能对吲哚途径具有核心调控作用;CYP2A6-1和CYP735A2等蛋白可能催化生成靛蓝、靛玉红、色胺酮前体的羟基化反应,对这两个蛋白与底物分子的结合进行分子对接建模,均显示对吲哚分子具有较好的结合力.本研究为后续开展菘蓝代谢调控和育种,以及开展吲哚类物质合成生物学研究提供候选基因.     

组合蛋白质工程和代谢工程设计全细胞催化剂合成靛蓝和靛玉红

从嗜高温放线菌Thermobifida fusca中分离得到的苯基丙酮单加氧酶主要催化芳香族化合物的Baeyer-Villiger氧化反应。对该酶的结构和功能进行研究时,发现位于底物结合口袋的Met446位点突变可以赋予突变酶催化C-H键活化的新功能,氧化吲哚合成靛蓝和靛玉红,但产量仅为1.89 mg/L。为了获得合成靛蓝和靛玉红的全细胞催化剂,直接补加吲哚并不能提高细胞合成效率,补加吲哚的前体物质L-色氨酸可以使细胞合成靛蓝和靛玉红的能力提高4.5倍,达到8.43 mg/L。为了进一步提高细胞的生物合成效率,通过代谢工程改造大肠杆菌的糖代谢途径,阻断葡萄糖异构酶基因pgi,使磷酸戊糖途径代替糖酵解途径成为葡萄糖的主要代谢通路,从而为细胞提供更多氧化吲哚所需的辅因子NADPH,导致细胞合成靛蓝和靛玉红的效率进一步提高3倍,达到25 mg/L。通过组合蛋白质工程和代谢工程设计全细胞催化剂不仅可以高效地合成靛蓝和靛玉红,而且设计理念为相关全细胞催化剂的开发提供了一种新的策略。     

根部淹水对菘蓝活性成分的影响

对菘蓝幼苗根系进行不同深度的淹水处理,采用HPLC法测定不同处理下菘蓝叶中靛蓝、靛玉红的含量。结果表明,淹水处理初期样品与对照相比,靛蓝的含量呈上升趋势,淹水后期急剧下降;靛玉红含量在淹水第1d急剧上升,之后随淹水时间延长不断降低。淹水深度越深对靛蓝、靛玉红含量影响越大。适当的淹水处理能诱导菘蓝叶中次生代谢产物靛蓝、靛玉红的合成与积累。该结果可为栽培菘蓝的质量控制和有效利用提供理论依据。     

青黛的化学成分

从十字花科植物靛青制成的青黛中,除了已知的靛蓝及靛玉红外,分离到四种化学成分,其中一种为色素成分,通过光谱分析及化学裂解,证明此成分的化学结构为6-吲羟-吲哚并[2,1b]喹唑啉酮-12,即青黛酮,并以吲哚酚与色胺酮缩合合成了青黛酮。其余三种成分鉴定为正-廿九烷、吲哚醌及色胺酮。     

NaCl胁迫过程中喷施ALA对菘蓝幼苗生长及主要活性成分含量的影响

以来源于安徽亳州和山西运城的菘蓝（Isatis indigotica Fort.）幼苗为实验材料,对100 mmol·L-1NaCl胁迫条件下喷施50.0、25.0、16.7、12.5和0.0 mg·L-15-氨基乙酰丙酸（ALA）后幼苗部分生长指标、叶片中靛蓝和靛玉红含量以及根中表告依春含量的变化进行了研究。结果表明：在100 mmol·L-1NaCl单一胁迫条件下,来源于安徽亳州的幼苗单株叶鲜质量和叶片中靛蓝和靛玉红含量均低于对照,单株根鲜质量、根冠比以及根中表告依春含量均高于对照;来源于山西运城的幼苗单株叶和根鲜质量、叶片中靛玉红含量和根中表告依春含量均低于对照,根冠比和叶片中靛蓝含量均高于对照。在NaCl胁迫过程中喷施ALA对菘蓝幼苗生长和有效成分的积累均有不同效应。其中,喷施25.0 mg·L-1ALA后安徽亳州产幼苗单株叶和根鲜质量均最高且显著高于NaCl单一胁迫处理组;喷施16.7 mg·L-1ALA后山西运城产幼苗单株叶和根鲜质量均较高但与NaCl单一胁迫处理组无显著差异。喷施50.0 mg·L-1ALA后安徽亳州产幼苗叶片中靛蓝含量最高（0.376 mg·g-1）,喷施25.0 mg·L-1ALA后其叶片中靛玉红含量最高（9.977 mg·g-1）,喷施16.7 mg·L-1ALA后其根中表告依春含量最高（0.229 mg·g-1）,且均显著高于NaCl单一胁迫处理组;喷施16.7 mg·L-1ALA后山西运城产幼苗叶片中靛蓝含量最高（0.282 mg·g-1）,喷施12.5 mg·L-1ALA后其叶片中靛玉红含量和根中表告依春含量均最高（分别为4.526和0.301 mg·g-1）,且均显著高于NaCl单一胁迫处理组。研究结果表明：喷施适宜浓度ALA能够有效减轻NaCl胁迫对菘蓝幼苗生长的影响、提高其体内药用活性成分的含量;总体上看,产自安徽亳州的菘蓝幼苗的耐盐性较强,且不同种源适宜的ALA浓度也有一定差异。     

HPLC法对比不同产地南板蓝根药材靛蓝、靛玉红含量的研究

目的:以高效液相色谱法对广东不同产地(广东高要、广东罗定、广东罗浮、广东鼎湖)南板蓝根药材及常用伪品(爵床科植物广西马蓝)的主要成分靛蓝、靛玉红进行含量测定,对比不同产地南板蓝根药材靛蓝、靛玉红含量的差别.方法:采用HPLC法建立南板蓝根药材中靛蓝与靛玉红的含量测定方法.色谱条件:Kromasil C18(4.6mm×250mm,5μm);以甲醇-水(75:25)为流动相;检测波长:290nm;流速:1.0ml/min.柱温:40℃.结果:①靛玉红、靛蓝线性范围分别为1.652～33.04μg/ml、1.284～25.68μg/ml.回收率分别为98.92%,RSD=1.52%,N=5(靛玉红);102.61%,RSD=1.28%,N=5(靛蓝);②广西马蓝中未检测出靛玉红、靛蓝.结论:该方法操作简便、准确快速、重现性好,可完善现行的南板蓝根的药材质量标准,有效控制南板蓝根药材的质量.     

西藏地区13种虾脊兰植物花部表型及花粉形态特征研究

该研究对西藏地区13种虾脊兰植物花部表型及花粉形态特征进行观察比较,目的为虾脊兰植物的分类鉴定和种间亲缘关系的确定提供新依据。研究结果显示:①13种虾脊兰在花颜色大致可分为黄色、黄绿色、黄褐色、粉红色4种,以粉红色的花居多;②13种虾脊兰均为总状花序,花各部位(萼片、花瓣、唇瓣、子房、柱头)的长、宽度测量指标均表现出不同程度的差异,萼片长、花瓣宽、唇瓣宽和柱头长4个测量指标的差异性最大,在各个种之间的P值结果表现出显著性差异水平;③这13种虾脊兰的花粉块均由成千上万个单粒花粉组成,未见到萌发孔结构,有些虾脊兰植物的花粉粒之间可以看到有明显黏结物质。花粉块大小不一,整体形状多呈卵圆形、长椭圆形和梨形;④不同种的虾脊兰花粉块表面形态特征有所差异,花粉粒纵向或横向排列堆积在一起,花粉粒形状也各不相同;⑤不同种虾脊兰的花粉粒表面纹饰特征区别明显,可划分为凹陷型、光滑型、小孔型和穿孔型。以上结果表明花部表型及花粉形态特征可作为13种虾脊兰植物分类鉴定的参考性状,对这13种虾脊兰植物亲缘关系的确定和系统分类有补充价值。     

菜粉蝶对两种迁地保护的兰科植物传粉和繁殖成功的作用

迁地保护是防止物种灭绝的有效措施。为了解在新栖息地兰科植物与昆虫之间的关系, 从而为兰科植物的保护提供借鉴, 作者对迁地栽培于深圳市全国兰科植物种质资源保护中心的本地种血叶兰(Ludisia discolor)和外来种银带虾脊兰(Calanthe argenteo-striata)的传粉系统和繁殖策略进行了观察和研究。血叶兰和银带虾脊兰都具有白色小花组成的花序, 且花有距。在开放栽培条件下, 菜粉蝶(Pieris rapae)是这两种兰科植物唯一有效的传粉者。菜粉蝶携带血叶兰和银带虾脊兰花粉块的部位分别是降落时平衡身体的左侧足部和取食花蜜的喙。两种兰科植物传粉效率较高, 花粉移走与沉降的比值分别为1.14 : 1和0.74 : 1。血叶兰的结实率在2007和2008两年间无显著差异, 且均高于银带虾脊兰。究其原因, 一方面是由于它为传粉者提供报酬; 另一方面是因为血叶兰的花期与菜粉蝶的盛发期相吻合, 有利于充分利用传粉者; 而银带虾脊兰的花期滞后于菜粉蝶的盛发期, 传粉者缺失使得部分花未能授粉。血叶兰和银带虾脊兰都是高度自交亲和的物种, 但不存在无融合生殖和自动自花授粉的现象; 自交和异交的繁殖成功率无显著差异。这两种兰科植物可能存在由传粉者导致的自交现象。血叶兰和银带虾脊兰在迁地栽培到兰科中心后生长良好, 每年花期都有自然结果的现象, 植株周围有新生幼苗的建立, 种群有逐渐扩大的趋势, 显示出迁地保护的可行性。可以预测若干年后, 这两个种在兰科中心具有建立稳定种群的可能性。     

云南种子植物分布新记录

报道了云南新记录种子植物10种7变种1变型,隶属于9科16属,即柔毛泡花树、山青木、白毛四照花、缙云四照花、沙梾、小叶臭味新耳草、纤枝兔儿风、小苦荬、兔儿风蟹甲草、华合耳菊、纤细半蒴苣苔、尾叶黄芩、长唇筒冠花、南川鼠尾草、短叶虾脊兰、带唇兰、类头状花序藨草和细杆筇竹等。     

缺氮和复氮对菘蓝幼苗生长及氮代谢的影响

对基质育苗后水培的菘蓝进行缺氮与复氮处理,分析其生长情况及氮代谢产物含量的变化,探讨缺氮和复氮对菘蓝幼苗生长及氮代谢的影响,以提高菘蓝产量和品质以及栽培过程中的氮素利用效率。结果显示:(1)正常供氮条件下,菘蓝幼苗的叶绿素含量、谷氨酰胺合成酶(GS)活性、硝态氮含量、靛玉红含量为最高,而其株高、主根直径、根的鲜重与干重、叶的鲜重与干重、根系活力均最小。(2)缺氮处理增加了菘蓝幼苗的主根直径和根干重,提高其根系活力和硝酸还原酶(NR)活性,促进游离氨基酸在叶中的积累;但降低了GS的活性,也降低了叶中硝态氮、可溶性蛋白、靛玉红及根中游离氨基酸的含量;缺氮对叶中靛蓝的含量无明显影响。(3)复氮处理增加了菘蓝幼苗的株高、主根长、根鲜重、叶鲜重、叶干重,提高了其根系活力,降低了NR和GS的活性;与对照相比,复氮降低了叶中硝态氮含量,提高了叶中可溶性蛋白、靛蓝及根中游离氨基酸的含量,但对叶中游离氨基酸和靛玉红含量影响较小。研究表明,缺氮后再复氮有利于菘蓝幼苗叶的生长,同时有利于增加其叶内靛蓝含量,从而提高其产量和品质。     

Tissue and intracellular localization of indican and the purification and characterization of indican synthase from indigo plants

Indican (indoxyl beta-D-glucoside) was found to accumulate only in green leaves of the indigo plant, and not in any other tissues. Comparisons of the indican content of protoplasts and vacuoles showed that indican was stored only in the vacuole of the cell. Indican content appeared and increased with the appearance and growth of leaves. In mature plants, the younger leaves contained larger amounts of indican than the older ones. Cell extracts of young leaves of indigo plant catalyzed the synthesis of indican from UDP-glucose and indoxyl. Indican synthase was extracted and purified from young leaves. The enzyme was separated into two fractions by anion-exchange chromatography. The enzyme in the fraction which was eluted by 0.1 M NaCl had a molecular weight of 53,000 by SDS-PAGE. Optimum pH of the enzyme was at about 10.0, indicating that the enzyme is likely localized in a different intracellular compartment from that of indican storage. The enzyme showed normal Michaelis-Menten kinetics and a K(m) value of 0.13 mM for UDP-glucose.     

夏播菘蓝不同居群干物质和活性成分积累特征

为研究夏播菘蓝不同栽培居群的最佳采收期,以来自于山西、安徽、甘肃、江苏和河南的5个栽培居群为材料,设置盆栽土培实验,于菘蓝生长第60天起,间隔10d采样,共采集6次样品,采用HPLC法测定其叶片、叶柄、根茎和根4个部位的靛蓝与靛玉红含量。结果表明:(1)不同栽培居群随着生长时间的延长,其叶片、叶柄、根茎及根的生物量持续增加,其中来自于山西居群的生长最佳,干物质积累量最大。(2)不同居群叶片及叶柄内靛蓝与靛玉红含量最大值出现在生长90~100d,即为叶内活性成分积累的高峰期。(3)综合分析其活性成分与干物质的积累量,各居群叶片靛蓝积累量最大值在生长90~100d,叶片、叶柄与根茎的靛玉红积累量也出现在90~100d。若以中国药典的靛玉红为质量控制指标,同时综合考察其生物量指标与活性成分积累量指标,夏季播种的来自于山西、安徽和河南的菘蓝居群最佳采收期为生长100d左右,而来自于甘肃与江苏居群的则以生长90d最佳。     

Beta-glucosidase-catalyzed hydrolysis of indican from leaves of Polygonum tinctorium

In this article, a HPLC method to identify and quantify the dyes and the indigo precursors produced in Polygonum tinctorium is described. Using this technique, indican has been positively identified in extracts of P. tinctorium. Our work with two cultivars of P. tinctorium has confirmed that the quantity of indican is dependent on the cultivars, harvest period, and age of the leaves. Two enzymes, Novozym 188 (cellobiase) and Novarom G (beta-glucosidase), are compared on the basis of their activities to hydrolyze the indican at several pH values. We observed that Novarom G is more active than Novozym 188 whatever the pH and that optimum pH of both enzymes for indican hydrolysis is 3. Liberated indoxyl can be oxidized in alkaline media and transformed into indigo and indirubin.     

Identification of an indigo precursor from leaves of Isatis tinctoria (Woad).

Indole is presumably a product of indole-3-glycerol phosphate catabolism in Isatis tinctoria. It is oxidized into indoxyl and stored in young leaves as indigo precursor. Further oxidation and dimerization of indoxyl produces indigoid pigments. In this work, we describe an HPLC method dedicated to the identification and quantification of indigoid pigments (indigo, indirubin, isoindigo and isoindirubin) and indigo precursors produced in I. tinctoria (Woad). This work, carried out with two cultivars of I. tinctoria, has confirmed that the quantity of indigo precursors is dependent on the species and the harvest period. In addition we have shown for the first time that young leaves of I. tinctoria, harvested in June contained a new indigo precursor in addition to isatan B (indoxyl-5-ketogluconate) and indican (indoxyl-beta-D-glucoside). We suggest the name "isatan C" for this new indigo precursor in I. tinctoria. Its chemical characteristics point to an dioxindole ester with PM of 395. We have shown that isatan C reacts with isatan B increasing the red pigment production.     

Formation of the indigo precursor indican in genetically engineered tobacco plants and cell cultures

The production of the blue dye indigo in plants has been assumed to be a possible route to the introduction of novel coloration into flowers or fibres. As the human cytochrome P450 mono-oxygenase 2A6 (CYP2A6) can form indigo in bacterial cultures, we investigated whether the expression of the corresponding cDNA in transgenic plants could lead to indigo formation. In a first attempt, we generated tobacco cell suspension cultures expressing the cDNA encoding human CYP2A6. Supplementation of the medium with indole led to the generation of indican (3-hydroxyindole-β- d -glucoside), a metabolite usually exclusively present in indigoferous dye plants. Hence, the recombinant CYP2A6 converted indole to the reactive metabolite 3-hydroxyindole (indoxyl), whereas rapid glucosylation is obviously conducted by ubiquitous plant glucosyl transferases (GTs). Interestingly, of nine additionally tested plant cell suspension cultures from various plant families, five were also capable of the formation of indican after indole supplementation, although this metabolism was more pronounced in transgenic tobacco cell suspension cultures expressing CYP2A6 cDNA. To evaluate whether indican or even indigo could be produced in whole plants, we generated transgenic tobacco plants harbouring active CYP2A6 together with an indole synthase (BX1) from maize. The genetically engineered tobacco plants accumulated indican, but did not develop a blue coloration. Although the de novo formation of indican in transgenic tobacco plants hampered indigo formation, it supports the contention that biosynthetic pathways can be efficiently mimicked by metabolic engineering.     

A new HPLC-ELSD method to quantify indican in Polygonum tinctorium L. and to evaluate beta-glucosidase hydrolysis of indican for indigo production

A method to quantify the indigo precursor indican (indoxyl-beta-D-glucoside) in Polygonum tinctorium L. has been developed. Plant material was extracted in deionized water, and indican was identified and quantified using high performance liquid chromatography (HPLC) coupled to an evaporative light scattering detector (ELSD). Results confirmed that with this method it is possible to measure indican content in a short time, obtaining reliable and reproducible data. Using this method, leaf indican content was quantified every 15 days during the growing season (from May to October) in P. tinctorium crops grown in a field experiment in Central Italy. Results showed that indican increased along the growing season until flowering and was positively affected by photosynthetic active radiation (PAR). Indican is naturally hydrolyzed by native beta-glucosidase to indoxyl and glucose, the indoxyl yielding indigo. The activity of two enzymes, sweet almond beta-glucosidase and Novarom G preparation, were compared with P. tinctorium native beta-glucosidase to evaluate indigo production. Results showed that the ability to promote indigo formation increased as follows: almond beta-glucosidase 相似文献   

The content of indigo precursors in Isatis tinctoria leaves--a comparative study of selected accessions and post-harvest treatments

We recently clarified the nature of indigo precursors in woad (Isatis tinctoria L.), by identifying the major indoxyl glycoside as isatan A (indoxyl-3-O-(6'-O-malonyl-beta-D-ribohexo-3-ulopyranoside)), and by correcting the structure of the related isatan B (indoxyl-3-O-beta-D-ribohexo-3-ulopyranoside). A quantitative densitometric assay for isatans A and B, and indican, was established and validated. HPTLC separation on silica gel was followed by densitometric analysis of indigoid pigments formed after treatment with dilute acid or base. The seasonal variation of indoxyl glycosides in woad leaves was investigated with first-year plants (rosette stage) of five defined I. tinctoria L. and one I. indigotica L. accessions. Isatan A content reached up to 7.6% of dry weight in I. tinctoria, and up to 21.8% in I. indigotica. The influence of various post-harvest treatments was studied. High concentrations of isatans A and B were found in freeze-dried leaf samples, whereas the content of indican was lowest. Conventional drying at ambient or 40 degrees C led to complete disappearance of isatans A and B. The concentration of indican, in contrast, was 3- to 5-fold higher in leaf samples submitted to drying at ambient and 40 degrees C, respectively.     

Isolation of a new potent cytotoxic pigment along with indigotin from the pathogenic basidiomycetous fungus Schizophyllum commune

An indole derivative, schizocommunin, was isolated along with indigotin (indigo), indirubin, isatin, and tryptanthrin, from the liquid culture medium in which a culture of Schizophyllum commune, isolated from the bronchus of a human patient with allergic bronchopulmonary mycosis, had been grown. The structure of schizocommunin was established by spectroscopic investigation. Schizocommunin showed the strong cytotoxicity against murine lymphoma cells. The assignments of the ¹H- and ¹³C-NMR signals of indigotin were also listed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Protein engineering of toluene ortho-monooxygenase of Burkholderia cepacia G4 for regiospecific hydroxylation of indole to form various indigoid compounds

Previous work showed that random mutagenesis produced a mutant of toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 containing the V106A substitution in the hydroxylase -subunit (TomA3) that changed the color of the cell suspension from wild-type brown to green in rich medium. Here, DNA shuffling was used to isolate a random TOM mutant that turned blue due to mutation TomA3 A113V. To better understand the TOM reaction mechanism, we studied the specificity of indole hydroxylation using a spectrum of colored TOM mutants expressed in Escherichia coli TG1 and formed as a result of saturation mutagenesis at TomA3 positions A113 and V106. Colonies expressing these altered enzymes ranged in color from blue through green and purple to orange; and the enzyme products were identified using thin-layer chromatography, high performance liquid chromatography, and liquid chromatography–mass spectroscopy. Derived from the single TOM template, enzymes were identified that produced primarily isoindigo (wild-type TOM), indigo (A113V), indirubin (A113I), and isatin (A113H and V106A/A113G). The discovery that wild-type TOM formed isoindigo via C-2 hydroxylation of the indole pyrrole ring makes this the first oxygenase shown to form this compound. Variant TOM A113G was unable to form indigo, indirubin, or isoindigo (did not hydroxylate the indole pyrrole ring), but produced 4-hydroxyindole and unknown yellow compounds from C-4 hydroxylation of the indole benzene ring. Mutations at V106 in addition to A113G restored C-3 indole oxidation, so along with C-2 indole oxidation, isatin, indigo, and indirubin were formed. Other TomA3 V106/A113 mutants with hydrophobic, polar, or charged amino acids in place of the Val and/or Ala residues hydroxylated indole at the C-3 and C-2 positions, forming isatin, indigo, and indirubin in a variety of distributions. Hence, for the first time, a single enzyme was genetically modified to produce a wide range of colors from indole.