花生二氢黄酮醇还原酶基因(DFR)的克隆及表达分析

二氢黄酮醇还原酶(dihydroflavonol 4-reductase,DFR)是植物花青素合成过程中的关键酶,催化二氢黄酮醇生成无色花青素。本研究利用花生(Arachis hypogae a L.)未成熟种子cDNA文库,通过大规模EST测序,从花生中克隆了DFR基因的全长cDNA序列。序列分析表明,花生DFR蛋白氨基酸序列与其他植物来源的DFR有很高的同源性。利用花生cDNA芯片和半定量RT-PCR方法对花生DFR基因表达模式的研究发现,DFR基因在果针中表达水平最高,其次是花,在根和叶中有微量表达;不同种皮颜色花生品种的种子中表达差异明显,随种皮颜色的加深,DFR基因表达增强,在中花9号黑色种子中表达量最高。对茎叶紫色花生种质材料的研究表明,在紫色组织中DFR基因表达较强。结果表明DFR表达量与花青素积累量呈正相关,说明DFR催化反应是花青素合成途径的关键步骤。     

马铃薯愈伤组织中色素含量与4个花色苷合成相关基因的表达差异

本研究从马铃薯（Solanum tuberosum cv．Chieftain）茎的愈伤组织中分离得到绿色、白色和红色3种愈伤组织,并利用鲜重法和分光光度法分别测量愈伤组织的生长量、叶绿素和花色苷的含量;通过半定量RT—PCR法分析4个花色苷生物合成相关基因的表达差异。结果表明：绿色愈伤组织生长最快,叶绿素含量最高,红色愈伤组织生长最慢,叶绿素含量最低;绿色和白色愈伤组织中几乎不含花色苷,红色愈伤组织中花色苷含量最高,达2．1OD513/mgFw;对CHS、F3H、DFR和F3’5’H四个花色苷生物合成相关基因表达分析发现,绿色和白色愈伤组织不合成花色苷可能与DFR基因不表达有关。本实验结果为进一步阐明花色昔生物合成机理和花色苷色素的生产应用提供一定的理论依据。     

干旱条件下DCMU对高表达转C4-pepc水稻的花青素合成基因及其相关信号的影响

为了揭示高表达转玉米C4-磷酸烯醇式丙酮酸羧化酶(PEPC,EC 4.1.1.31)基因水稻(PC)在耐旱中光合与花青素调节途径的内在联系,本研究以PC和未转基因野生型原种(WT)的水培苗为试验材料,在4~5叶期,通过50μmol·L-1光合抑制剂DCMU[3-(3’,4’-dich-lorophenyl)-1,1-dimethyl-urea]预处理1 h,观察其在12%PEG-6000模拟干旱处理下的表现。结果表明,在模拟干旱条件下,DCMU预处理使两种供试材料相对含水量显著下降,且PC相对含量显著高于WT;干旱处理下,两种材料的花青素含量显著升高,DCMU和干旱处理使两种材料的花青素含量下调,且PC水稻中始终伴随着较高的花青素含量。光合数据表明,与单独12%PEG-6000处理相比,DCMU联合12%PEG-6000处理显著抑制了两种水稻材料的净光合速率、气孔导度、胞间CO2含量及羧化效率,但PC的各指标显著高于WT。同时,DCMU联合12%PEG-6000处理显著下调两种供试材料的内源蔗糖含量,但PC中蔗糖含量显著高于WT。进一步研究发现PC中更高的蔗糖含量与花青素合成有关转录因子b HLH(Os B1,Os B2)、R2R3-MYB(Os C1)、COP1(constitutively photomorphogenic 1)、HY5(elongated hypocotyl 5)更高的转录水平同步,下游花青素合成相关基因Os PAL、Os CHI、Os CHS、Os F3H、Os F3’H、Os DFR、Os ANS的表达量增加。PC水稻可能通过诱导NO和Ca2(10)感受干旱信号,参与转录因子的调节,进而参与花青素合成基因的调控,合成较多的花青素,增强PC水稻对干旱逆境的响应,增强保水能力,最终表现耐旱。     

NO和H2O2依赖Ca2+/CaM信号调控马铃薯花青素积累

以不同块茎颜色马铃薯品种陇薯8号、陇薯7号、LC310-2和山东彩肉为材料,研究外源NO、H2O2、NO+ H2O2处理对马铃薯块茎花青素合成及其信号物质Ca2+-ATPase和CaM的影响.结果表明:外源NO和H2O2处理后,4个基因型马铃薯薯皮和薯肉花青素积累,Ca2+-ATPase活性升高,CaM含量增加,其效果为NO+ H2O2＞H2O2＞NO＞水(CK),并且花青素的积累量与Ca2+-ATPase活性和CaM含量均呈正相关,其平均决定系数达0.9194和0.8859.说明NO和H2O2对马铃薯花青素合成具有一定的促进作用,而细胞Ca2 +/CaM信号发挥了NO和H2O2对马铃薯花青素合成诱导的信号调节.     

水稻花青素合成相关基因的时空表达研究

水稻花青素的生物合成受很多因素的影响,最根本的影响因素是结构基因和调控基因的表达水平不同,导致色素积累的差异性.本文通过半定量PCR方法研究一些结构基因和调节基因的时空表达差异性.发现OsPAL,OsCHS和OsCHI为花青素的基本表达基因,在叶片、茎、花期及花后1～2周的种子中均有表达.OsF3H,OsF3’H和OsDFR不在茎中表达,在叶片、花期和花后1～2周内均有表达,且在花后1～2周内,其表达量与时间成正比.OsANS在黑米和紫叶水稻的叶片和花中有少量表达,在花后1～2周内,仅在黑米中表达,且表达量与时间成正比.OsB1和OsB2特异性地在紫叶水稻的叶片中表达,OsC1也特异地在花期时表达.说明水稻不同部位的花青素积累受控于不同的基因,具有一定的时空表达特异性.     

AtDREB1A基因过量表达对马铃薯盛花期生理生化指标的影响

以马铃薯品种陇薯3号及其转AtDREB1A基因株系T1为研究对象,在干旱胁迫条件下,研究了AtDREB1A基因过量表达对盛花期生理生化指标的影响。分析表明,在不同程度的干旱胁迫下,转基因株系T1盛花期叶片的超氧化物歧化酶(SOD)活性及脯氨酸(Pro)含量均高于同期非转基因陇薯3号对照,而丙二醛(MDA)含量和相对电导率均低于陇薯3号。以上结果初步表明,AtDREB1A基因的过量表达明显提高了马铃薯对干旱的抗性。     

葡萄风信子二氢黄酮醇4-还原酶基因(DFR)的克隆与表达分析

葡萄风信子(Muscari armeniacum)是一类重要的球根花卉,其花色呈现特殊的蓝色。花青素是影响葡萄风信子蓝色形成的重要物质,与植物生长过程及人类/动物饮食密切相关。有关花青素生物合成途径的研究已较为成熟,但由于葡萄风信子基因资源缺乏,其花色形成的机理目前尚不清楚。二氢黄酮醇4-还原酶(dihydroflavonol 4-reductase,DFR)是植物花青素合成中的重要酶。本研究采用RACE技术从葡萄风信子花瓣中克隆到两条DFR基因(MaDFR2a和MaDFR2b),GenBank登录号分别为KJ619963和KJ619964。序列分析表明,MaDFR2a和MaDFR2b全长1 392 bp,包含一个长度为76 bp的5'非翻译区和215 bp的3'非翻译区,均包括一个1 101 bp的完整开放阅读框(open reading frame,ORF),编码366个氨基酸,氨基酸相似性为98%,与其他植物的DFR蛋白同源性较高。聚类分析表明,葡萄风信子DFR最先与单子叶植物聚为一类。生物信息学分析表明,两个DFR蛋白可能为亲水性蛋白,均具有两个跨膜区,无信号肽。亚细胞定位分析显示,两个DFR基因均定位于整个细胞中。α-螺旋和无规则卷曲是两个DFR蛋白的主要二级结构元件。MaDFR2a和MaDFR2b编码的蛋白三级结构非常相似,中间形成一个裂沟,可以进行催化反应。花青素含量测定显示,着色的花器官中花青素含量较高,根、茎、叶以及未着色的花蕾中几乎检测不到花青素。荧光定量PCR分析发现,MaDFR2a和MaDFR2b在葡萄风信子花组织中表达较高,根、茎和叶中微量表达;MaDFR2a和MaDFR2b均在未着色的花蕾时期开始表达,随着花色加深,在完全着色的花中表达最高,此后逐渐降低,但MaDFR2b在未着色的花蕾期和完全着色的花中表达相对较高。研究结果表明,DFR可能是影响蓝色葡萄风信子形成的重要基因。     

植物二氢黄酮醇-4-还原酶基因的研究进展

二氢黄酮醇-4-还原酶(DFR)是花青素生物合成途径中的关键酶,在花色的修饰中起重要作用,目前,已从多种植物中分离得到。从DFR基因的结构和作用机制、底物特异性、时空表达模式及花色修饰等方面进行了概括和总结,为DFR基因的进一步研究和利用提供理论依据。     

马铃薯花青素转录激活基因(stmyc)的克隆及表达分析

花青素（anthocyanin）为糖苷类物质,属于类黄酮色素。在花青素生物合成途径中,调控基因主要编码R2R3-MYB、bHLH和WD40转录因子,共同调控花青素合成的结构基因的表达,进而控制花青素在植物中的时空表达和沉积的模式（Holton and comish,1995）。本研究以紫色马铃薯为材料,克隆花色素合成的转录激活基因stmyc并研究其表达模式,为阐明马铃薯花色素生物合成和沉积机制提供依据。     

TRV介导的葡萄叶片VvANR基因瞬时表达分析

为建立葡萄叶片TRV-VIGS系统,分析验证VvANR基因功能,本研究以烟草脆裂病毒(Tobacco rattle virus,TRV)介导基因沉默表达载体pTRV2-ANR,采用真空侵染和主叶脉针孔注射法分别侵染葡萄幼嫩、成熟叶片,观察表型并测定原花青素含量,实时荧光定量PCR测定被侵染叶片中VvANR及其相关基因表达。结果表明,真空侵染的幼嫩、成熟叶片分别于第6、第7天出现漂白,侵染15 d后,幼嫩叶片几乎全部漂白,成熟叶片大面积漂白。主叶脉针孔注射侵染的幼嫩、成熟叶片分别于第17、第19天出现漂白;侵染25 d后,幼嫩和成熟叶片发生全叶漂白,且漂白均沿主叶脉逐渐扩展至整个叶面;对照均未发生漂白现象。pTRV2-ANR侵染后叶片中原花青素含量极显著降低,VvANR基因表达量极显著降低,且原花青素生物合成相关基因VvLAR1、VvLAR2、VvMYBPA1、VvMYBPA2、VvUFGT表达量显著降低,除VvUFGT表达量呈显著差异外,其余均呈极显著差异;而VvANS、VvDFR表达量轻微上调,且与对照差异不显著。本研究建立了葡萄叶片VIGS体系,为基因功能快速验证提供了新方法;同时,揭示了葡萄叶片中原花青素含量、VvANR表达及与相关基因的关系,本研究为完善葡萄原花青素积累机制奠定了一定的理论基础。     

Anthocyanin absorption and antioxidant status in pigs

The effect of a simultaneous intake of food or flavonoids on anthocyanins absorption and antioxidant status in pigs was investigated. Twelve male pigs at 27.1 +/- 0.7 kg BW fitted with jugular venous cannulae were maintained in individual metabolic crates. The animals were each given one of three dietary treatments in random order: blackcurrant powder (BC) to give a dose of 100 mg total ACNs/kg BW mixed either with water and sugar (Diet A), cereal (Weet-Bix), milk, and sugar (Diet B), or cereal, milk, sugar, and an additional flavonol (rutin, approximately 100 mg/kg BW) (Diet C). The four major anthocyanins of BC, delphinidin-3-glucoside, delphinidin-3-rutinoside, cyanidin-3-glucoside, and cyanidin-3-rutinoside, were identified and quantified by HPLC-PDA in all three diets. In the pig plasma, four peaks with a reversed pattern to those of anthocyanins in the BC extract were detected. The total amount of anthocyanins absorbed was not significantly different between the three diets, but the rate of absorption and subsequent decline was slower following administration of diet B and C than diet A. All three diets increased antioxidant capacity when measured by the FRAP assay but not when measured by the ORAC and non-protein ORAC assay. However, the increase was delayed and did not appear until 4 h after ingestion, at a time when plasma anthocyanin levels had returned to baseline. The present study demonstrates that the simultaneous intake of food or other flavonoids delays the absorption profile for anthocyanins. Our results also suggest that the increase in antioxidant capacity is not due to dietary anthocyanins but may be due to metabolites that result from anthocyanin consumption.     

Anthocyanin composition of wild bananas in Thailand

Anthocyanins were isolated from male bracts of 10 wild species of bananas (Musa spp. and Ensete spp.) distributed in Thailand. Six major anthocyanin pigments were identified by high performance liquid chromatography (HPLC), mass spectrometry (MS), and tandem mass spectrometry (MS/MS). They are delphinidin-3-rutinoside (m/z 611.2), cyanidin-3-rutinoside (m/z 595.8), petunidin-3-rutinoside (m/z 624.9), pelargonidin-3-rutinoside (m/z 579.4), peonidin-3-rutinoside (m/z 608.7), and malvidin-3-rutinoside (m/z 638.8). On the basis of the types of pigment present, the wild bananas can be divided into 5 groups. The first group comprises M. itinerans, Musa sp. one, Musa sp. two, and M. acuminata accessions, which contain almost or all anthocyanin pigments except for pelargonidin-3-rutinoside, including both nonmethylated and methylated anthocyanins. The second group, M. acuminata subsp. truncata, contains only malvidin-3-rutinoside while the third group, M. coccinea, contains cyanidin-3-rutinoside and pelargonidin-3-rutinoside. The forth group, M. acuminata yellow bract and E. glaucum do not appear to contain any anthocyanin pigment. The fifth group consists of M. balbisiana, M. velutina, M. laterita, and E. superbum which contain only nonmethylated anthocyanin, delphinidin-3-rutinoside, and cyanidin-3-rutinoside. Total anthocyanin content in the analyzed bracts ranged from 0-119.70 mg/100 g bract fresh weight. The differences in the type of anthocyanin and variation in the amounts present indicate that wild bananas show biochemical diversity, which may be useful for identifying specific groups of bananas or for clarifying the evolution of flavonoid metabolism in each banana group.     

红叶葡萄品种叶片花色苷种类的鉴定与分析

为解决葡萄园中由葡萄修剪产生的大量叶片残留问题,提高修剪产生的叶片的利用价值,本试验以8612、玫瑰香、红鸡心3个红叶葡萄品种和葡萄园皇后、玫瑰露、秋红3个绿叶葡萄品种不同发育时期的叶片为研究对象,利用MATLAB软件对整张叶片的图像数据进行提取,计算色差值;利用超高效液相-质谱法(LC-MS)检测花色苷的成分和含量,探讨不同发育时期叶片中花色苷不同组分的变化规律。结果表明,不同发育时期的红色和绿色叶片色差指数L^*、a^*、b^*变化趋势明显不同,共检测到18种花色苷组分,包括花青素类(4种)、甲基花青素类(4种)、花翠素类(4种)、甲基花翠素类(2种)和二甲基花翠素类(4种)。以上5类花色苷在红色叶片中均被检测到,绿叶品种中未发现花翠素类和甲基花翠素类花色苷。花色苷定量结果显示,红色叶片不同发育时期花色苷含量为123.468~855.001 mg·100g^-1,绿色叶片花色苷含量为4.407~44.517 mg·100g^-1。甲基化类花色苷占比随叶片发育均逐渐增大。花色苷酰化修饰类型分析结果发现,香豆酰化类型花色苷含量高于其他酰化类型花色苷,在花色苷总含量中所占比例较高,而阿魏酰化和糖酰化类花色苷含量非常少。色差和不同类型花色苷成分的相关性分析结果表明,红色叶片的色差指数与更多类型的花色苷含量存在相关关系。红色葡萄叶片中花色苷种类丰富、含量较高,是花色苷类化合物的潜在来源,具有很大的利用价值。本研究通过对叶片中花色苷成分和含量进行了详细调查,为今后葡萄园中叶片的加工再利用提供了依据。     

Anthocyanin and ascorbic acid degradation in sonicated strawberry juice

Strawberry juice samples were sonicated at amplitude levels ranging from 40 to 100% at a constant frequency of 20 kHz for treatment times (2-10 min) and pulse durations of 5 s on and 5 s off. Sonication was found to reduce anthocyanin and ascorbic acid contents by 3.2 and 11%, respectively, at the maximum treatment conditions. Response surface methodology (RSM) based on a two-factor, five-level central composite design was employed to determine the effect of amplitude level and treatment time on anthocyanins (P3G), ascorbic acid (AA) content, and color values (L*, a*, and b*). The model predictions for the selected nutritional and quality parameters were closely correlated to the experimental results. RSM was demonstrated to be an effective technique to model the effect of sonication on strawberry juice quality while minimizing the number of experiments required.     

Anthocyanin antioxidant activity and partition behavior in whey protein emulsion

The antioxidant activities of anthocyanins and anthocyanin fractions isolated from blackcurrants, raspberries, and lingonberries were investigated in whey protein-stabilized emulsion. The extent of protein oxidation was measured by determining the loss of tryptophan fluorescence and formation of protein carbonyl compounds and that of lipid oxidation by conjugated diene hydroperoxides and hexanal analyses. The antioxidant activity of berry anthocyanins increased with an increase in concentration. Blackcurrant anthocyanins were the most potent antioxidants toward both protein and lipid oxidation at all concentrations due to the beneficial combination of delphinidin and cyanidin glycosides. Most berry anthocyanins (69.4-72.8%) partitioned into the aqueous phase of the emulsion, thus being located favorably for antioxidant action toward protein oxidation. The presence of the lipid decreased the share of anthocyanin in the aqueous phase. Thus, the structure of food affects the antioxidant activity by influencing the partitioning of the antioxidant.     

Anthocyanin Content and Composition in Winter Blue Barley Cultivars and Lines

This study focuses on the examination of the total anthocyanin content (TAC) and spectrum of winter barley cultivars with blue caryopsis. Preparation of grain samples was simplified, and extraction was performed with 85% methanol acidified by 1% trifluoroacetic acid. Compared with our previous tests performed with 1% hydrochloric acid in aqueous methanol, the extraction process resulted in higher total anthocyanin content with more acylated derivatives. The TAC measured by spectrophotometry varied in two blue Hungarian cultivars and two lines from 46.9 to 84.4 mg/kg. The anthocyanin spectrum was analyzed by electrospray ionization tandem mass spectrometry. The predominant anthocyanins were delphinidin‐3‐malonylglucoside and cyanidin‐3‐malonylglucoside, followed by delphinidin‐3‐glucoside and cyanidin‐3‐glucoside. The caryopsis retains the anthocyanins in long‐term storage. The TAC values of the crops over the last 10 years were definable retroactively. The relative abundance of the main anthocyanin components in grains of different harvest years (2000 and 2010) was similar. Statistical analysis revealed a relatively strong negative correlation (r = –0.7439, P < 0.05) between two variables: TAC and rainfall during the grain‐ripening period.     

基因工程及花色素合成途径在花色改良中的研究进展

随着经济的发展,转基因技术在花卉中的应用也越来越关键,通过基因工程使花色发生变化已经在商业中运用。生物碱类、胡萝卜素和类黄酮化合物等一类活性物质共同作用使植物的花色更具观赏性,目前,在花色改良上应用较多的是类黄酮色素,其研究机理也较为清楚。本文主要介绍了F3＇5＇H（类黄酮-3＇,5＇-羟基化酶）和F3＇H（类黄酮-3＇-羟基化酶）的生物合成途径,阐述了基因工程技术对花色修改的作用,综述了目前花卉转基因工程的研究现状和花色素合成的主要途径及影响因素,同时简单介绍了目前改良花色的几种策略和就其存在的问题、发展前景做出了讨论。     

Anthocyanin color behavior and stability during storage: effect of intermolecular copigmentation

Intermolecular copigmentation reactions are significantly responsible for the manifold color expression of fruits, berries, and their products. These reactions were investigated with five anthocyanins and five phenolic acids acting as copigments. The stability of the pigment-copigment complexes formed was studied during a storage period of 6 months. The study was conducted using a UV-visible spectrophotometer to monitor the hyperchromic effect and the bathochromic shift of the complexes. The greatest copigmentation reactions took place in malvidin 3-glucoside solutions. The strongest copigments for all anthocyanins were ferulic and rosmarinic acids. The immediate reaction of rosmarinic acid with malvidin 3-glucoside resulted in the biggest bathochromic shift (19 nm) and the strongest hyperchromic effect, increasing the color intensity by 260%. The color induced by rosmarinic acid was not very stable. The color intensity of pelargonidin 3-glucoside increased greatly throughout the storage period with the addition of ferulic and caffeic acids.     

Anthocyanin transformation in Cabernet Sauvignon wine during aging

Anthocyanins in Cabernet Sauvignon grapes and wines were elucidated by HPLC-MS/MS. Major anthocyanins in Cabernet Sauvignon grape extract are malvidin 3-O-glucoside and malvidin 3-O-acetylglucoside. In matured wine, anthocyanins are transformed to anthocyanin-vinyl derivatives, ethyl bridged anthocyanin-flavanol adducts, and anthocyanin-flavanol adducts. The major anthocyanin pigments are malvidin 3-O-glucoside-pyruvate, malvidin 3-O-acetylglucoside-pyruvate, malvidin 3-O-coumaroylglucoside-pyruvate, malvidin 3-O-glucoside-4-vinylphenol, malvidin 3-O-acetylglucoside-4-vinylphenol, and malvidin 3-O-coumaroylglucoside-4-vinylphenol. The presence of syringetin 3-O-glucoside and syringetin 3-O-acetylglucoside has been established for the first time in grape and wine.     

Impact of Irrigation Thresholds on Total Anthocyanin Content in Cranberries

The impact of irrigation thresholds based on soil water potentials on total anthocyanin content (TAcy) of cranberry fruit was studied at two sites. Two methods based on spectrophotometric measurements were compared: the acidic solvent extraction (ASE) method and the pH-differential method. TAcy was greater by 21–29 percent when irrigation was managed using a wetter threshold, ?5.5 and ?6.0 kPa, in comparison with a drier threshold, from ?6.5 to ?10.0 kPa. The pH-differential method resulted in significantly greater TAcy measurements than the ASE method, although the ASE method was faster and simpler to use.