观赏羽衣甘蓝BoDFR基因的克隆及表达分析

为揭示羽衣甘蓝二氢黄酮醇4 还原酶 (DFR)基因调控花青素合成的功能,该研究对不同叶色羽衣甘蓝的叶片花青素含量进行测定,根据结球甘蓝DFR序列信息,利用 RT PCR 技术克隆羽衣甘蓝BoDFR基因并进行实时荧光定量表达分析。结果表明: BoDFR的cDNA全长为1 158 bp,编码385个氨基酸,其蛋白相对分子质量为42 925.06 Da,预测亚细胞定位为细胞质;蛋白质二级结构分析表明α 螺旋和无规则卷曲为DFR 蛋白的主要二级结构元件。序列比对显示 DFR 蛋白具有 NADPH 结合位点和底物结合位点,属于NADB Rossmann 超基因家族。系统进化分析表明,BoDFR与结球甘蓝(Brassica oleracea var. capitata)DFR亲缘关系最近。花青素含量测定显示,紫叶羽衣甘蓝叶片中花青素含量最高,粉叶羽衣甘蓝含量较高,而白叶羽衣甘蓝叶片中检测不到花青素。实时荧光定量 PCR 分析表明,BoDFR基因表达量与花青素含量高低一致,其中紫叶羽衣甘蓝叶片中BoDFR基因的表达量最高,而白叶羽衣甘蓝心叶中仅微量表达。     

枫香秋季变色期叶色变化及其生理基础

为探究秋季枫叶呈色的关键生理因素,该文以转色期叶色为绿色、黄色和红色的枫香单株为试材,研究了L^*、a^*、b^*值变化与叶片色素、可溶性糖及可溶性蛋白质含量变化的相关性。结果表明:(1)在变色期,3种色彩枫香叶片叶绿素a、叶绿素b、总叶绿素和类胡萝卜素均大量降解,花色素苷不同程度积累。(2)绿色叶单株叶绿素和类胡萝卜素始终保持较高含量,花色素苷含量上升4.2倍,叶片内色素含量比值始终保持稳定; 黄色叶单株叶绿素和类胡萝卜素含量最低,花色素苷含量上升4.4倍,b^*值与叶绿素含量极显著负相关,与类胡萝卜素含量显著负相关,与花色素苷/类胡萝卜素含量比值极显著正相关; 红色叶单株叶绿素和类胡萝卜素含量略高于黄色叶单株,花色素苷含量上升27.2倍,a^*值与叶绿素含量、类胡萝卜素含量极显著负相关,与花色素苷含量显著正相关,与色素含量比值无显著相关性。(3)红色叶单株具有较高的可溶性糖含量和可溶性蛋白质含量。因此,在枫香叶片变色期,保持较高的叶绿素和类胡萝卜素含量,维持色素含量比值稳定使叶片呈现绿色; 叶绿素和类胡萝卜素的大量降解,以及花色素苷/类胡萝卜素含量比值的升高使叶片呈现黄色; 叶绿素的降解和花色素苷的大量合成使叶片呈现红色。     

金花茶类黄酮糖基转移酶基因的克隆和功能初步研究

类黄酮糖基转移酶(UDP flavonoid glycosyltransferase, UFGT)催化黄酮醇、花青素等形成稳定的糖苷,是黄酮醇苷、花青素苷合成的最后一步反应的关键。该研究以金花茶的花瓣为材料,采用PCR扩增的方法,获得了2个金花茶转录组中筛选到的类黄酮糖基转移酶基因。结果显示：(1)CnUFGT14基因(登录号为MT370521)全长1 562 bp,开放阅读框1 380 bp,编码459个氨基酸;CnUFGT15基因(登录号为MT370520)全长1 546 bp,开放阅读框1 368 bp,编码455个氨基酸;两个蛋白序列均具有UFGT蛋白特有的 PSPG 保守区域。(2)系统进化树分析发现,CnUFGT14和CnUFGT15分别与茶树UFGT78A14和UFGT78A15亲缘关系最近。(3) 荧光定量PCR分析发现,CnUFGT14基因的表达量与多种多酚组分的含量呈正相关,CnUFGT15基因的表达量与花瓣黄酮醇、多酚等的含量相关性不显著。(4)亚细胞定位研究发现,CnUFGT14、CnUFGT15蛋白在细胞核膜、细胞质、细胞膜部位均呈现明显的定位。(5)叶盘法转化烟草发现,CnUFGT14基因表达量较高的转基因株系中总多酚含量及多种多酚组分含量升高,而CnUFGT15基因的转基因株系中黄酮、多酚组分变化不显著。研究表明,CnUFGT14基因具有促进多酚合成的作用,而CnUFGT15基因对类黄酮通路不具有明显作用。     

葡萄风信子MaGT1基因的克隆及其表达分析

该研究以葡萄风信子(Muscari ameniacum)‘亚美尼亚’为试材,克隆了花青素合成途径过程中的类黄酮糖基转移酶基因MaGT1(GenBank登录号MK652470)。该基因开放阅读框全长1 338 bp,编码445个氨基酸,预测蛋白分子量为49.301 kD,理论等电点为5.40。结构分析显示,MaGT1蛋白具有PSPG保守结构域、UDP 糖基转移酶家族结构域和UDP 葡萄糖醛酸基/葡萄糖基转移酶保守域(UDPGT)。进化分析表明,MaGT1蛋白与油棕、海枣、葡萄亲缘关系相近,聚类于类黄酮糖苷糖基转移酶类分支,以UDP 葡萄糖/鼠李糖为主要糖供体。花青苷含量测定显示,花青苷仅在葡萄风信子着色的花中积累,在根、鳞茎和叶以及未着色的花蕾(S1)中几乎检测不到花青苷,且随着花的发育,花青苷含量不断增加,并在衰败期(S5)达到最高。荧光定量PCR分析表明,MaGT1基因的表达具有显著的时空特异性,并在花中优势表达,而在根、鳞茎和叶片中微量表达;在花发育不同阶段,MaGT1基因的表达量随着花发育不断增加,并在盛花期达到峰值。研究表明,MaGT1蛋白催化反应是花青素合成途径中的重要修饰步骤。该研究结果为进一步分析MaGT1基因在葡萄风信子花青素合成和调控中的功能提供了依据。     

甘薯IbGL3的克隆和表达分析

紫色甘薯富含花青素,具有较高的食用和药用价值。花青素的生物合成受到结构基因和调节基因的控制。bHLH(basic helix-loop-helix protein)转录因子能够调节多个花青素结构基因的表达,在花青素生物合成途径中具有重要的调控作用,但目前在甘薯中还没有关于bHLH调控花青素生物合成的相关报道。为进一步了解IbGL3基因在甘薯花青素生物合成的功能和作用机理,该研究根据甘薯转录组数据,利用RT-PCR技术在甘薯中克隆了一个2 120 bp的bHLH基因IbGL3,该基因包含一个1 878 bp的开放阅读框,编码625个氨基酸,蛋白质分子量69.08 kD,理论等电点(pI)5.20。IbGL3蛋白和其他植物中类黄酮合成相关的bHLH蛋白具有较高的同源性,都包含保守的MIR区、bHLH结构域和ACT类似结构域。系统发育进化树分析结果显示,IbGL3与其他植物类黄酮相关bHLH蛋白聚为一类,属于Ⅲf 亚类成员。表达结果显示,IbGL3基因在紫色甘薯中的表达量最高,在浅紫色甘薯中的表达量次之,在白色甘薯中表达量最低, 与花青素积累正相关,因此推测其在甘薯花青素生物合成途径中具有重要的调控作用。     

矮牵牛PhTPS5基因的克隆与表达分析

海藻糖 6 磷酸合成酶（Trehalose 6 phosphate synthase）基因TPS是海藻糖生物合成途径中的关键基因之一。该研究从矮牵牛 (Petunia hybrida)中分离了TPS5的同源基因PhTPS5。该基因开放阅读框（ORF）为2 595 bp,编码864个氨基酸。推测PhTPS5蛋白的分子式为C⁴³⁶³H⁶⁸²⁵N¹¹⁷³O¹²⁸⁹S³⁷。组织特异性表达分析显示：PhTPS5基因在根中表达量最高,叶片中的表达量最低;去顶6 h能够显著促进PhTPS5基因的表达,但24 h后表达量明显下降;去顶后施加生长素则能够有效抑制去顶对PhTPS5基因表达的调节;施加细胞分裂素6 h后PhTPS5基因的表达水平显著上调,但随着处理时间的增加,其表达水平有所下降。该研究为进一步揭示TPS途径在矮牵牛分枝发育中的调控机制奠定了理论基础。     

用基因芯片检测DPYD等位基因在受试人群中的发生频率

二氢嘧啶脱氢酶基因（DPYD基因）所编码的二氢嘧啶脱氢酶（DPD酶）是氟化嘧啶类抗肿瘤药物代谢的主要限速酶,其活性存在显著的个体差异,并因此影响药物的疗效和毒副作用.大部分编码低/无活性酶的突变型等位基因是由于基因中的单核苷酸多态性（single nucleotide polymorphism,SNP）造成的,检测这些SNPs是预测患者对药物的反应和实现个体化给药方案的基础.制备并优化了用于检测DPYD基因中6个已知SNPs所编码的等位基因（DPYD^*2,^*3,^*4,^*5,^*9,^*12）的基因芯片,建立了该芯片的基因分型标准.并利用该芯片检测了肿瘤患者（112例）、肾病患者（83例）和健康者（45例）中DPYD突变型等位基因的发生频率.在受试人群中,突变型等位基因DPYD^*5和DPYD^*9平均发生率分别为32.08%和11.25%,未发现DPYD^*2,^*3,^*4,^*12突变型等位基因.而且以上单碱基突变的发生率在肿瘤患者、肾病患者和健康者间以及男性、女性肿瘤患者间无显著性差异,表明其与疾病的发生或性别无显著性关联.对20例标本的基因分型结果采用直接测序法进行验证,19例基因芯片分型结果与直接测序法结果相一致.DPYD^*5、DPYD^*9突变型等位基因在受试人群中具有较高的发生率.利用基因芯片能够对其实现快速准确的检测.     

黑果枸杞LrTTG1基因的克隆及表达分析

以黑果枸杞为材料,利用RT PCR和RACE技术克隆了花青素合成相关基因LrTTG1（GenBank登录号为MH633481）。序列分析表明,LrTTG1基因cDNA全长1 453 bp,包含1 029 bp开放阅读框,编码342个氨基酸,含有5个WD40重复基序。同源比对结果表明,LrTTG1与茄子SmTTG1的氨基酸序列相似性较高,达到83.73%。qRT PCR分析显示,LrTTG1基因在茎、叶、花、青果、紫果和黑果中均有表达,且在青果中的表达水平（最高）约为黑果（最低）的4倍;紫外胁迫下LrTTG1基因的表达随胁迫时间的延长呈先降低后升高的变化趋势。花青素含量分析表明,黑果的花青素含量最高（11.3 mg/g）,分别约为紫果（ 1.2 mg/g）和青果（0.53 mg/g）含量的9.4倍和21.3倍。研究表明,随着黑果枸杞果实的发育,LrTTG1基因的表达量呈现下降趋势,而花青素的含量则呈上升趋势,两者呈负相关关系;推测LrTTG1基因在黑果枸杞花青素合成中可能具有重要的调节作用。     

不同产地浙贝母生物碱含量及其合成相关基因表达研究

为了探究浙贝母(Fritillaria thunbergii)药效成分积累量与生物碱合成相关基因表达水平之间的关系,该研究采用UPLC-MS、qPCR技术分别测定不同产地11个样品中总生物碱(贝母素甲和贝母素乙之和)含量和3个参与生物碱合成途径相关基因(HMGR、FPS和DXR)的表达量,同时运用生物统计学方法分析成熟期鳞茎生物碱含量与各基因表达量之间的相关性。结果表明:不同产地浙贝母成熟期鳞茎总生物碱含量存在显著差异(P<0.05),为0.2105% ～ 0.4612%; HMGR和FPS基因在盛花期组织表达、盛花期至成熟期鳞茎表达变化趋势同生物碱含量变化趋势基本一致; DXR基因在成熟期鳞茎中表达量最高,盛花期组织表达、盛花期至成熟期鳞茎表达变化趋势同生物碱含量变化趋势大体不一致; HMGR、FPS基因表达量分别与贝母素甲、贝母素乙和总生物碱含量呈显著或极显著正相关性(P<0.05或P<0.01),以FPS基因表达量与生物碱含量相关系数为最高,相关系数分别为0.672,0.631,0.664,DXR基因与生物碱含量呈低度相关性。由此可以推断,生物碱的积累受MVA途径中HMGR、FPS基因协同调控或者修饰作用明显,受MEP途径中DXR基因调控作用不明显。     

梅花新品种‘治章骨红重翠’跨品种群特性机制探究

‘治章骨红重翠’梅是一个具有跨品种群特性的梅花新品种,它同时具有朱砂和绿萼品种群的典型特征,其萼绿、花白、木质部淡暗紫色,但其机制并不清楚。本研究对‘治章骨红重翠’梅、‘豫西朱砂’梅、‘豫西变绿萼’梅表型、花色苷含量和花青素合成通路相关基因的表达量进行测定,结果表明,花瓣、萼片和枝内新生木质部的红色程度与总花色苷含量呈正相关。MYBɑ1、MYB1、bHLH3是影响这3个品种花部与木质部呈现红色的关键转录因子基因,转录因子促进结构基因F3′H、DFR、ANS和UFGT高表达,从而促进红色性状的产生。综合分析后推测,‘治章骨红重翠’梅花瓣白色源于矢车菊合成通路上其他分支FLS、F3′5′H、LAR和ANR基因高表达,谷胱甘肽转移酶(glutathione transferase, GST)基因GST低表达;萼片绿色源于F3′H、DFR和ANS基因低表达;木质部红色源于ANS、UFGT基因高表达。本研究对‘治章骨红重翠’梅跨品种群特性作出初步解释,为梅花花色与木质部颜色的分子育种提供了参考。     

六种藓类植物茎的比较解剖学研究

通过对6种藓类植物,即褶叶青藓(Brachythecium salebrosum(Web.et Mohr.)B.S.G.)、湿地匐灯藓(Plagiomnium acutum(Lindb.)Kop.)、侧枝匐灯藓(Plagiomnium maximoviczii(Lindb.)Kop.)、大凤尾藓(Fissidensnobilis Griff.)、大羽藓(Thuidium cymbifolium(Doz.et Molk.)B.S.G.)和大灰藓(Hypnum plumaeforme Wils.)嫩茎和老茎的石蜡切片和显微观察发现,同一藓类植株的嫩茎和老茎,茎结构稳定,不同种藓类植物茎横切面具有不同特征.植物体茎横切面形状、表层细胞的层数、细胞大小和细胞壁厚薄、皮层细胞大小和形状、中轴的有无以及比例等特征可以作为藓类植物的分科分类依据之一.     

Photoregulation of seed germination of wild-type and of an aurea-mutant of tomato

Seed germination of an aurea mutant of tomato ( Lycopersicon esculentum Mill.) is promoted by continuous irradiation with red, far-red or long-wavelength far-red (758 nm) light as well as by cyclic irradiations (5 min red or 5 min far-red/25 min darkness). Far-red light applied immediately after each red does not change the germination behaviour. Seed germination of the isogenic wild-type, cv. UC-105, is promoted by continuous and cyclic red light while it is inhibited by continuous and cyclic far-red light and by continious 758 nm irradiation. Far-red irradiation reverses almost completely the promoting effect of red light. The promoting effect (in the aurea mutant) and the inhibitory effect (in the wild-type) of continuous far-red light do not show photon fluence rate dependency above 20 nmol m⁻² s⁻¹. It is concluded that phytochrome controls tomato seed germination throgh low energy responses in both the wild type and the au mutant. The promoting effect of continuous and cyclic far-red light in the au mutant can be attributed to a greater sensitivity to P_fr.     

Cause of Senescence of Nine Sorts of Flowers

The levels of endogenous phytohormones and respiratory rate in nine sorts of flowers such as Cymbidium faberi Rolfe, Nopalxochia ackermannii Kunth and others were investigated both at full bloom and senescence and meanwhile the effect of exogenous phytohormones on prolonging the blossoms and promoting ethylene production were tested. There is a high content of endogenous ethylene in all the long-lived flowere, about 3–16 folds higer than the short-lived ones. There is a high level of ABA at full blooming flowers of short-lived flowers, in which there is no or only some cytokinins in it, but the ratio of CTK (6BA+zeatin)/ABA is smaller(l.7). The endogenous ABA reached a much higher level at senescence in all nine sorts of flowers, so it is reasonable to consider that it is ABA which plays an important role of regulation in controlling flower's senescence. There is a much higher level of GA3 and zeatin in the long-lived flowers which is not demonstrated in the shortlived ones. The respiratory rate is one of the factors controtling the longevity of flowers, but it does not play a decided role. Application of 6BA and zeatin prolongs distinctly orchid’s longevity, however exogenous IAA through the promotive action on ethylene production, evidently extends the longevity of the flowers of the Nopalxochia ackermannii Kunth.     

龙胆科药用植物化学成分的研究现状

龙胆科植物在我国的分布范围很广,且多数为药用植物,其多数种属的药用植物,至今其化学成分尚未被系统研究。综述了目前龙胆科药用植物的化学成分的研究现状及一般提取方法,对近年来发现的环烯醚萜及裂环烯醚萜类化合物进行了总结,为本科药用植物的更深入研究提供了参考。     

Interconnection of parameters of regulation system of lipid peroxidation and of morphophysiological parameters of mouse liver

A complex analysis of seasonal fluctuations of the mean group parameters of the system of regulation of lipid peroxidation has been performed in liver of Balb/c mice. Association of lipid characteristics and morphophysiological parameters is studied in the Balb/c mouse liver. An inter-connection is revealed between the liver index and the amount of lysoforms of phospholipids, the scale and character of the interconnection differing essentially depending on proportion of phos-phatidylcholine in mouse liver phospholipids.     

The effects of glutamine of the maintenance of embryogenic cultures of Cryptomeria japonica

Summary Embryogenic tissues of sugi (Cryptomeria japonica) were induced on a modified Campbell and Durzan (CD) medium containing 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 600 mg l⁻¹ glutamine, and subcultured in the medium of the same composition for over 1 yr. This resulted in a mixed culture of embryogenic and non-embryogenic cells. When embryogenic cells were isolated and cultured independently, their capacity to form embryogenic aggregates was lost. Thus, the non-embryogenic cells present within a mixed culture system were essential to the formation of embryogenic aggregates. When embryogenic tissues were isolated and cultured independently on a high glutamine-containing (2400 mg l⁻¹) medium, dry weights and endogenous levels of glutamine increased, and the tissue could generate a large number of embryogenic aggregates. Amino acid analysis of embryogenic and non-embryogenic cells from the maintenance culture indicated a higher level of glutamine was present in the latter. The high endogenous level of glutamine in the non-embryogenic portion of mixed cell masses may be the supplier of glutamine for maintaining the embryogenic property of the tissues.     

Specificity of action of piperidylcholine derivatives as substrates of cholinesterases of various origin

The review deals with study of enzymologic properties of a novel highly specific acetylcholinesterase substrate, N-(β-acetoxyethyl) piperidinium iodomethylate (“piperidylcholine”), and its 30 derivatives that were tested as effectors of cholinesterases of mammals and various species of Pacific squids. It was proven for the first time that responsible for specificity of action was structure of cyclic ammonium grouping of the alcohol part of molecule of the ester substrate. Analysis of specificity is performed based on enzymatic hydrolysis parameters—activity of catalytic center of cholinesterases and bimolecular constant of the reaction rate that are determined at optimal and low substrate concentrations. Among the specially synthesized group of thioester compounds there is revealed one more highly specific acetylcholinesterase substrate—N-(β-acetoxyethyl) piperidinium.     

Physiological characteristics of various species of strains of carboxydobacteria

Seven strains of aerobic carbon monoxide-oxidizing bacteria (carboxydebacteria) when growing on CO as sole source of carbon and energy had doubling times which ranged from 12–42 h. The activity profiles obtained after discontinuous sucrose density gradient centrifugation indicated that the CO-oxidizing enzymes are soluble and the hydrogenases are membrane-bound in all strains examined. The CO-oxidizing enzymes of Pseudomonas carboxydohydrogena, Pseudomonas carboxydoflava, Comamonas compransoris, and the so far unidentified strains OM2, OM3, and OM4 had a molecular weight of 230,000; that of Achromobacter carboxydus amounted to 170,000. The molecular weights of the CO-oxidizing and H₂-oxidizing enzymes turned out to be identical. The cell sonicates were shown to catalyze the oxidation of both CO and H₂ with methylene blue, thionine, phenazine methosulfate, toluylene blue, dichlorophenolindophenol, cytochrome c or ferricyanide as electron acceptors. Methyl viologen, benzyl viologen, FAD⁺, FMN⁺, and NAD(P)⁺ were not reduced. The spectrum of electron acceptors was identical for all strains tested. Neither free formate, hydrogen nor oxygen gas were involved in the CO-oxidation reaction. Methylene blue was reduced by CO at a 1:1 molar ratio. The results indicate that CO-oxidation by carboxydobacteria is catalyzed by identical or similar enzymes and that the reaction obeys the equation CO+H₂OCO₂+2H⁺+2e^- as previously shown for Pseudomonas carboxydovorans.Dedicated to Otto Kandler remembering almost three decades of enjoyable cooperation     

Modulation of allostery of pyruvate kinase by shifting of an ensemble of microstates

Since the introduction of the concepts of allostery about four decades ago, much advancement has been made in elucidating the structure-function correlation in allostery. However, there are still a number of issues that remain unresolved. In this review we used mammalian pyruvate kinase (PK) as a model system to understand the role of protein dynamics in modulating cooperativity. PK has a triosephosphate isomerase (TIM)(α/β)₈ barrel structural motif. PK is an ideal system to address basic questions regarding regulatory mechanisms about this common (α/β)₈ structural motif. The simplest model accounting for all of the solution thermodynamic and kinetic data on ligand-enzyme interactions involves two conformational states, inactive E^T and active E^R. These conformational states are represented by domain movements. Further studies provide the first evidence for a differential effect of ligand binding on the dynamics of the structural elements, not major secondary structural changes. These data are consistent with our model that allosteric regulation of PK is the consequence of perturbation of the distribution of an ensemble of states in which the inactive E^T and active E^R represent the two extreme end states. Sequence differences and ligands can modulate the distribution of states leading to alterations of functions. The future work includes: defining the network of functionally connected residues; elucidating the chemical principles governing the sequence differences which affect functions; and probing the nature of mutations on the stability of the secondary structural elements, which in turn modulate allostery.