不同色彩矮牵牛DFR基因的克隆与生物信息学分析

二氢黄酮醇4-还原酶基因(DFR)是花色素合成途径中的一个关键基因。以新疆种植的白、红和蓝色矮牵牛为试验材料,通过同源克隆的方法从花中克隆到3个完整的DFR基因的全长编码序列(CDS),与已知的矮牵牛DFR基因(GenBank登录号:X15537)序列的相似性分别为97.79%、96.59%和97.99%,分别命名为PhDFR1,PhDFR2和PhDFR3;3个基因编码380个氨基酸,同已知矮牵牛DFR基因编码的蛋白(GenBank登录号:CAA33544)的同源性分别是95.53%、94.21%和95.79%;生物信息学分析表明,3个蛋白均具有NADB-Rossmann家族中高度保守的NADPH结合位点、底物特异性结合位点。3个矮牵牛品种DFR都不具有信号肽,为亲水蛋白,定位于细胞质的可能性最高;均具有两个跨膜结构,α-螺旋和β-折叠是3个DFR的主要二级结构元件,并且形成了β-α-β-α-β的Rossmann折叠,整本上呈对称分布。利用同源建模分析3个DFR蛋白与已知葡萄的DFR晶体结构有很高的相似性。系统进化树分析表明,PhDFR1、PhDFR2、PhDFR3与已知矮牵牛DFR蛋白亲缘关系最近。     

不同栽培区域和年份的红皮砂梨果皮着色特性

该研究以红皮砂梨品种‘八月红’和‘红香酥’为试材,比较了不同栽培地区和年份的红皮砂梨着色规律、花色素苷组分及含量,以及相关代谢酶的变化。结果显示:（1）‘八月红’和‘红香酥’果皮花色素苷含量变化规律由其品种特性决定,同一品种的果皮花色素苷绝对含量受不同栽培区域和年份的影响,其组成和含量变化趋势不受影响;但不同品种的花色素苷含量变化以及着色规律有所不同。（2）红色砂梨果皮着色主要与矢车菊-3-半乳糖苷含量变化一致,且随着梨果实的发育和着色加深,果实中UDP-葡萄糖:类黄酮-3-O葡萄糖苷转移酶（UFGT）、二氢黄酮醇-4-还原酶（DFR）活性呈上升趋势,而苯丙氨酸解氨酶(PAL)活性随着果实发育而下降。（3）相关性分析表明,两品种梨果实花色苷含量与其UFGT、DFR活性呈显著或极显著正相关关系,而与PAL活性相关性不一致。研究表明,梨果皮中的UFGT、DFR是影响红皮砂梨着色的重要酶。     

棕色棉DFR基因的克隆与生物信息学分析

花色素苷是影响花色的主要色素,二氢黄酮醇4-还原酶(DFR)基因是花色素苷生物合成途径的关键酶基因。通过同源克隆策略,以新彩棉6号(XC-6)纤维的RNA以及DNA为模板克隆得到GhDFR基因的CDS全长编码序列及带有内含子的基因组序列,并进行了生物信息学分析。序列分析结果显示,该基因含有6个外显子,5个内含子结构,其cDNA包含一个1 020 bp的开放阅读框,编码355个氨基酸,其氨基酸序列包含具有高度保守性的NADP(H)的结合位点以及底物特异性结合位点。推定的GhDFR蛋白质分子量为39.65 kD,等电点为5.67。该蛋白氨基酸序列同毛果杨、葡萄、天竺葵等物种DFR蛋白显示出较高同源性,而系统进化分析结果表明其与天竺葵、芍药DFR亲缘关系较近。氨基酸序列分析预测表明,GhDFR基因所编码的蛋白不具备信号肽区段,无明显跨膜区域,不属于分泌蛋白,可能为亲水性蛋白,定位于细胞质的可能性最高,其主要二级结构元件为α-螺旋和无规则卷曲。GhDFR属于NADB-Rossmann superfamily。     

二氢黄酮醇4-还原酶基因(DFR)与花色的修饰

二氢黄酮醇4-还原酶(DFR)是花色素苷生化合成途径中的一个多种植物中分离到了DFR基因.文章介绍DFR同源基因的结构与时空表达特性、构建的系统进化树所体现的部分单子叶与双子叶植物间亲缘关系与进化差异以及DFR的转基因研究进展.     

花色素苷对拟南芥耐盐性的影响

为探讨花色素苷在盐胁迫中的防御作用及其机制,以模式植物拟南芥(Arabidopsis thaliana)花色素苷合成途径相关基因缺失突变体(DFR基因缺失突变体tt3,CHS基因缺失突变体tt4,CHS、DFR基因双缺失突变体tt3tt4)及其野生型(WT)为材料,采用叶绿素荧光和超氧阴离子(O_2·~–)组织定位等方法,分析了tt3,tt4,tt3tt4和WT对盐胁迫处理的生理响应。结果表明,盐胁迫下3种缺失突变体叶片花色素苷含量的增加显著低于野生型,与WT相比,叶绿素荧光参数Fv/Fm、Yield、ETR、q P和NPQ下降较快,膜渗漏率升高显著,叶片O_2·~–积累程度为tt3tt4tt3/tt4WT。这表明花色素苷在植物抵御盐胁迫过程中起着重要作用,它可能是作为渗透调节剂及抗氧化剂来增强植物的耐盐性。因此,花色素苷含量可以作为筛选耐盐作物的指标。     

外源ABA对低温不同光周期下四季秋海棠叶片花色素苷的诱导机理

以‘超级奥林匹克’四季秋海棠(‘Super Olympia’ Begonia semperflorens)为材料,于常温（25 ℃/15 ℃）等日照条件下用0、5、10、50、100、500 μmol/L脱落酸(ABA)和低温（15 ℃/6 ℃）不同光周期下用10 μmol/L ABA分别喷施各处理植株,对不同处理下植株的色素含量、内源激素含量及其酶活性进行比较分析,探讨ABA对其叶片花色素苷合成的调控作用及其机理。结果显示：（1）常温等日照条件下,四季秋海棠叶片在5和10 μmol/L ABA处理后的第3~5天有明显变红趋势,且花色素苷含量和内源ABA含量显著增加,而内源赤霉素（GA）含量的下降幅度较为显著,相应的花色素苷合成关键酶和运输酶活性也显著提高。（2）低温条件下,四季秋海棠叶片花色素苷积累量与光周期密切相关,短日照处理的花色素苷积累量最大并显著高于等日照和长日照,但等日照与长日照下花色素苷积累量无显著差异;外源10 μmol/L ABA处理均可显著提高低温不同光周期下四季秋海棠叶片的花色素苷含量。（3）外施10 μmol/L ABA增加了低温下长日照和等日照处理中DFR（二氢黄酮醇 4 还原酶）还原反应中H供体NADPH的含量,促使DFR和UFGT（糖苷转移酶）的活性上调进而增加了花色素苷的含量;外施ABA处理均能够提高低温不同光周期处理组的内源ABA含量,降低内源GA的含量,与花色素苷的生成量相一致。研究表明,外源ABA能够通过调节花色素苷合成的关键酶来影响花色素苷的合成,外施适宜浓度ABA能够促进四季秋海棠叶片花色素苷的积累,可用于实际生产中的叶片着色调控管理。     

增强UV-B对矮牵牛花瓣中生理生化物质变化的影响

比较研究了普通光照(CK)和增强UV-B处理条件下,粉红色矮牵牛(Petunia hybrida)花瓣中花色素苷、辅色素、丙二醛和还原糖的含量以及苯丙氨酸解氨酶(PAL)活性的变化.结果表明,UV-B照射处理组花瓣中花色素苷、辅色素和还原糖含量均高于对照(CK),PAL活性增强,叶面积、叶宽和周长均小于对照组,MDA含量则低于对照组.综合分析表明,粉红色矮牵牛承受增强UV-B处理有一个阈值,大致为960.5 kJ?m-2;并且花色素苷和辅色素的作用不同,花色素苷以抗氧化为主,而辅色素以对UV-B的屏蔽作用为主.     

矮牵牛新种质的花色表型及花色素分析

以27个上海交通大学自育矮牵牛新种质为研究材料,对花色这一重要观赏性状及其花色素进行了系统研究。用RHSCC比色和色差仪测色方法描述了矮牵牛的花色表型,通过特征显色反应初步判断了矮牵牛的花色素类型,以标准曲线法和pH示差法等方法测定了矮牵牛3类花色素的含量。研究表明：这27个矮牵牛种质的花色可归于5个色系,以紫红色和红色为主;矮牵牛花色在CIELab表色系统中分布较广,而且不同色系花色参数的区分度较大。矮牵牛花瓣中含有类黄酮和花色苷,不含或含少量类胡萝卜素。13个被测种质的花瓣类黄酮含量在2.5～12.2 mg·/g–1 ·FW之间,花色苷含量在0.08～3.88 mg·g–1 FWmg/g·FW之间,而类胡萝卜素在矮牵牛花瓣中含量很低,远远低于类黄酮含量,在7个被测种质中,最高仅为0.216 mg·g–1 FWmg/g·FW,最低为0.004 mg·g–1 FWmg/g·FW。以上结果显示,5个色系矮牵牛所含花色素种类不尽相同,含量也有明显差异,其中紫红色系和红色系花瓣大多不含或含极少量类胡萝卜素,黄色系、白色系和紫色系花瓣的类黄酮含量较高,紫色系和紫红色系花瓣花色苷含量较高。     

"彩色马铃薯"块茎花色苷分子结构研究进展

“彩色马铃薯”是指块茎的“皮”和／或“肉”为红、紫、蓝或橙色的马铃薯,其块茎“皮”和“肉”变化多端的着色模式源于花色苷的积累,块茎各种颜色在根本上由花色素决定。在“彩色马铃薯”块茎中已发现6种花色素,即矮牵牛色素、花葵素、锦葵色素、芍药色素、花青素和花翠素;不同颜色块茎所含的花色素种类不同,同一颜色块茎所含花色素种类也可能不同;紫色块茎所含的花色素种类最为多样化。“彩色马铃薯”块茎的各种花色素一般在C3位经过氧一糖苷键实现1个芸香糖基取代,在苷元的C5位,要么以氧．糖苷键实现单葡萄糖基取代,要么不发生取代。“彩色马铃薯”块茎花色苷常在花色素C3位二糖取代基上或在C5位的单糖取代基上进一步发生反式单酰基取代,实现酰基取代的酚酸多为对香豆酸,其次为阿魏酸和咖啡酸。“彩色马铃薯”块茎矮牵牛素、锦葵色素、花葵素和芍药色素的对香豆酸酰化衍生物的惯用名分别为“petanin”,“malvanin”,“pelanin”和“peonanin”。本文可以为“彩色马铃薯”块茎颜色呈现的机理探索及其花色苷的分子结构鉴定提供参考。     

不同颜色果袋对‘云红梨2号’果皮色泽形成的影响

研究了不同颜色果袋对‘云红梨2号’果实着色的影响,比较了不同套袋处理下果皮外观着色、叶绿素、类黄酮、总酚、花色素苷含量以及花色素苷合成相关酶活性的差异.结果表明: 发育期的黑暗处理有利于解袋后梨果皮着色;不同套袋处理中,采前解袋自然光照射下梨果皮中花色素苷含量最高,着色最好,白色纸袋次之.不同套袋处理显著影响果皮中叶绿素、类黄酮、总酚和花色素苷含量,从而影响梨果皮的外观色泽.不同套袋处理的花色素苷合成酶活性差异显著;相关性分析表明,果皮中花色素苷含量与二氢黄酮醇-4-还原酶(DFR)和类黄酮3-O-葡萄糖基转移酶(UFGT)活性呈显著正相关,而与苯丙氨酸解氨酶(PAL)活性相关性不显著.     

不同危险分层急性肺栓塞患者D-二聚体与纤维蛋白原比值、中性粒细胞与淋巴细胞比值、白蛋白的变化及其与预后的关系研究

摘要 目的：探讨不同危险分层急性肺栓塞（APE）患者D-二聚体与纤维蛋白原比值（DFR）、中性粒细胞与淋巴细胞比值（NLR）、白蛋白（Alb）的变化及其与预后的关系。方法：选择2019年3月至2021年12月我院收治的APE患者154例作为APE组,根据《肺血栓栓塞症的诊断与治疗指南(2015)》分为低危组48例、中危组69例和高危组37例,另选择同期我院体检健康志愿者40例作为对照组,比较各组DFR、NLR、Alb水平。根据不同预后将APE患者分为存活组125例,死亡组29例,比较两组DFR、NLR、Alb水平。应用受试者工作特征（ROC）曲线分析DFR、NLR、Alb对APE预后的预测价值。结果：APE组DFR、NLR显著高于对照组,Alb水平显著低于对照组（P<0.05）。随着危险分层增加,APE患者DFR、NLR逐渐升高,Alb水平逐渐降低,不同危险分层APE患者DFR、NLR、Alb水平比较有统计学意义（P<0.05）。死亡组DFR、NLR显著高于存活组,Alb水平显著低于存活组（P<0.05）。ROC曲线分析显示,DFR、NLR、Alb对APE死亡预测具有较高的敏感度、特异度,其中DFR、NLR、Alb联合检测对APE死亡预测的曲线下面积（AUC）、敏感度、特异度最高。结论：APE患者DFR、NLR异常升高,Alb异常降低与APE危险分层增加及不良预后相关,DFR、NLR、Alb联合检测对APE患者预后不良的预测价值更高。     

An in vitro model for the growth and analysis of chronic wound MRSA biofilms

Aims: To develop an in vitro model (Colony/drip‐flow reactor – C/DFR) for the growth and analysis of methicillin‐resistant Staphylococcus aureus (MRSA) biofilms. Methods and Results: Using the C/DFR model, biofilms were grown on the top of polycarbonate filter membranes inoculated with a clinical isolate of MRSA, placed on absorbent pads in the DFR and harvested after 72 h. The biofilms varied from 256 to 308 μm in thickness with a repeatability standard deviation of 0·22. Testing of antimicrobial agents was also performed where C/DFR biofilms were grown in parallel with conventional colony biofilms. A saline solution (control), 1% silver sulfadiazine solution, and 0·25% Dakin’s solution were used to treat the biofilms for 15 min. Microscopic evaluation of biofilm morphology and thickness was conducted. The Dakins solution in both models produced statistically significantly higher log reductions than silver sulfadiazine treatment. Conclusions: The C/DFR biofilms were thick and repeatable and exhibited higher resistance to Dakins solution than the treated colony biofilms. Significance and Impact of the Study: The C/DFR can be used as a tool for examining complex biofilm physiology as well as for performing comparative experiments that test wound care products and novel antimicrobials.     

CHEMICAL ANALYSIS OF TOAD EGG-JELLY IN RELATION TO ITS 'SPERM-CAPACITATING' ACTIVITY

In an attempt to characterize a factor in anuran egg-jelly that is essential for fertilization, dejellied, non-fertilizable eggs of the toad, Bufo bufo , were inseminated in the following jelly preparations: jelly solubilized by KCN followed by dialysis (Dialyzed jelly: DJ), jelly solubilized by ultraviolet irradiation (UVJ), a diffusible factor released from jelly coat into deionized water (DF), the dialyzable fraction of DF (DFD), and the non-dialyzable fraction of DF (DFR). It was found that all the preparations except DFR are active in supporting the fertilization of dejellied eggs. DFD is thermo-stable, and characterized by a rise in pH accompanying increase in concentration. DF obtained from Rana japonica also capacitated the fertilization of dejellied Bufo eggs.
Chemical analyses indicated that DJ, UVJ, DF and DFR contain various amounts of fucose, hexoses, hexosamines, and proteins. Sialic acid was present in DJ and UVJ, but not in DF. In DFD, only hexoses and proteins were detectable to a measurable degree. A salient feature of the paper chromatographic analyses was the predominance in DFD of an unspecified reducing sugar which was found in common in all the preparations with fertilization-supporting activity. Gel-filtration in combination with bioassay for fertilization led to the isolation of the active substance, which had a molecular weight of less than 500, and was characterized by a basic nature and the presence of a reducing sugar.
The possible importance in fertilization of this small molecular weight jelly component is stressed, together with the suggestion that the component represents some terminal group of the jelly macromolecule in either diffusible or non-diffusible form.     

Flavonoid synthesis in Petunia hybrida: partial characterization of dihydroflavonol-4-reductase genes

In this paper we describe the organization and expression of the genes encoding the flavonoid-biosynthetic enzyme dihydroflavonol-4-reductase (DFR) in Petunia hybrida. A nearly full-size DFR cDNA clone (1.5kb), isolated from a corolla-specific cDNA library was compared at the nucleotide level with the pallida gene from Antirrhinum majus and at the amino acid level with enzymes encoded by the pallida gene and the A1 gene from Zea mays.The P. hybrida and A. majus DFR genes transcribed in flowers contain 5 introns, at identical positions; the three introns of the A1 gene from Z. mays coincide with first three introns of the other two species. P. hybrida line V30 harbours three DFR genes (A, B, C) which were mapped by RFLP analysis on three different chromosomes (IV, II and VI respectively).Steady-state levels of DFR mRNA in the line V30 follow the same pattern during development as chalcone synthase (CHS) and chalcone flavanone isomerase (CHI) mRNA. Six mutants that accumulate dihydroflavonols in mature flowers were subjected to Northern blot analysis for the presence of DFR mRNA. Five of these mutants lack detectable levels of DFR mRNA. Four of these five also show drastically reduced levels of activity for the enzyme UDPG: flavonoid-3-O-glucosyltransferase (UFGT), which carries out the next step in flavonoid biosynthesis; these mutants might be considered as containing lesions in regulatory genes, controlling the expression of the structural genes in this part of the flavonoid biosynthetic pathway. Only the an6 mutant shows no detectable DFR mRNA but a wild-type level for UFGT activity. Since both an6 and DFR-A are located on chromosome IV and DFR-A is transcribed in floral tissues, it is postulated that the An6 locus contains the DFR structural gene. The an9 mutant shows a wild-type level of DFR mRNA and a wild-type UFGT activity.     

Molecular analysis of herbivore-induced condensed tannin synthesis: cloning and expression of dihydroflavonol reductase from trembling aspen (Populus tremuloides)

In order to study condensed tannin synthesis and its induction by herbivory, a dihydroflavonol reductase (DFR) cDNA was isolated from trembling aspen (Populus tremuloides). Bacterial overexpression demonstrated that this cDNA encodes a functional DFR enzyme, and Southern analysis revealed that DFR likely is a single-copy gene in the aspen genome. Aspen plants that were mechanically wounded showed a dramatic increase in DFR expression after 24 h in both wounded leaves and unwounded leaves on wounded trees. Feeding by forest tent caterpillar (Malacosoma disstria) and satin moth (Leucoma salicis) larvae, and treatment with methyl jasmonate, all strongly induced DFR expression. DFR enzyme activity was also induced in wounded aspen leaves, and phytochemical assays revealed that condensed tannin concentrations significantly increased in wounded and systemic leaves. The expression of other genes involved in the phenylpropanoid pathway were also induced by wounding. Our findings suggest that the induction of condensed tannins, compounds known to be important for defense against herbivores, is mediated by increased expression of DFR and other phenylpropanoid genes.     

A comprehensive analysis of six dihydroflavonol 4-reductases encoded by a gene cluster of the Lotus japonicus genome

Dihydroflavonol 4-reductase (DFR) is the first committed enzyme of the anthocyanin and condensed tannin pathways. Several DFR cDNAs have been cloned, and different specificities of DFR isozymes in the substrate hydroxylation patterns have been reported, but only fragmentary knowledge of DFR gene organization is available. Reported here is a comprehensive analysis of DFRs of a model legume, Lotus japonicus. A total of five DFR genes were found to form a cluster within a 38 kb region in the L. japonicus genome, whereas six cDNAs, including two splicing variants resulting from a transversion at a splicing acceptor site, were cloned. All the genes were expressed, with different organ specificities, in the mature plant. Three of the DFR proteins heterologously expressed in Escherichia coli showed catalytic activity, and their substrate preferences agreed with the variation of a specific active site residue (Asp or Asn) reported to control the specificity. The hydroxylation patterns of anthocyanidins and condensed tannin units in the stems did not reflect the substrate specificity of the expressed isozymes, implying complex regulation mechanisms in the biosynthesis. The two splicing variants and one DFR with Ser at the specificity-controlling position failed to show the activity, but a revertant protein replacing the unusual splicing restored the activity. The phylogenetic tree, constructed with known DFR sequences, showed evolutionary divergence of some of the DFR genes prior to the plant speciation. This work affords the basis for genetic and biochemical studies on the diversity of DFR and the flavonoid products.     

Characterization of dihydroflavonol 4-reductases for recombinant plant pigment biosynthesis applications

Anthocyanins are colorful plant pigments with promising applications as pharmaceuticals and colorants. In order to engineer efficient pigment biosynthesis in Escherichia coli, the activities of various dihydroflavonol 4-reductases (DFRs) were characterized for the three primary dihydroflavonol substrates. The biochemical assays demonstrated variable DFR activities for dihydroflavonol with one B-ring hydroxyl group, the precursor of pelargonidin derivatives. In contrast, dihydroflavonols with two and three B-ring hydroxylation were metabolized with comparable efficiency. Furthermore, the catalysis of DFR for the secondary substrates, flavanones, also depended on the number of B-ring hydroxyl groups. Engineering the expression of the DFR clones together with plant-specific 4-coumaroyl:CoA ligase, chalcone synthase, chalcone isomerase, and flavanone 3-hydroxylase in E. coli resulted in the synthesis of pelargonidin at various levels, from p-coumaric acids. The identification of a robust DFR from this study can also be used for engineering recombinant synthesis of other bioactive flavonoids, such as flavan-3-ols.     

蓝粒小麦花青素生物合成途径中的二氢黄酮醇4-还原酶基因的分子克隆

蓝色色素在蓝粒小麦种子糊粉层中的生物合成途径的分子生物学机制至今仍不清楚.应用RT-PCR和RACE方法从蓝粒小麦正在发育的种子中克隆到一个编码二氢黄酮醇4-还原酶的基因(DFR).推测其为花青素生物合成途径中的一个关键基因,且与蓝粒小麦中蓝色色素形成密切相关;其开放阅读框编码一个包含354个氨基酸残基的多肽,与一些从其他植物中已克隆到的DFR有很高的同源性:大麦(94%)、水稻(83%)、玉米(84%).从长穗偃麦草(2n=70)、蓝粒小麦、浅蓝粒小麦自交产生的白粒后代小麦以及中国春的基因组中分别分离到一个全长DFR序列.经聚类分析表明DFR cDNA核甘酸序列与从中国春基因组中克隆的DFR具有100%的同源性,且与长穗偃麦草、蓝粒小麦、白粒小麦基因组中分离的DFR均有很高的同源性.4个DFR基因组DNA均含有3个内含子,且它们之间的差异主要在内含子区,表明该基因在进化上很保守.经Southern杂交分析,DFR在小麦中至少有3～5个拷贝,不同小麦材料间未见明显差异,但与长穗偃麦草有明显差异,属于一个DFR超基因家族.Northern分析表明该DFR在蓝粒和白粒种子的不同发育时期的表达存在明显差异,都在开花后大约18 d表达最强,在同一时期的蓝白种子中,DFR在蓝粒种子中的表达量高于白粒.DFR转录本在小麦和长穗偃麦草的幼叶中积累多,但在芽鞘中的表达显著低于幼叶中;在小麦的根和长穗偃麦草的发育种子中均未检测到DFR的表达.推测蓝粒小麦中可能存在调控DFR在蓝粒小麦中表达的调控基因,类似于玉米花青素合成途径中的调节基因.