高等植物二氢黄酮醇4-还原酶基因研究进展

花青素苷是影响植物花瓣呈色的重要色素,而花色是决定花卉观赏价值和商业价值的一个重要因素。在花青素苷的生物合成过程中,二氢黄酮醇4-还原酶（DFR）是花青素苷生物合成下游途径中的第一个关键的酶。因此,DFR在高等植物花色的形成过程中发挥极其重要的作用,是形成花青素苷的一个非常重要的调控点。DFR对3种二氢黄酮醇底物具有选择特异性,但决定DFR底物特异性的分子机制目前仍不十分清楚。该文简单概述了花青素苷生物合成途径及其转录调控机制,并结合作者的工作重点综述了DFR的底物特异性以及克隆的DFR基因在植物基因工程中的应用。     

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18d表达最强,在同一时期的蓝白种子中,DFR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。     

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.     

七彩红竹二氢黄酮醇4-还原酶基因IhDFR1的克隆及表达分析

二氢黄酮醇4-还原酶（DFR）是植物花色素苷合成途径中的关键酶,在植物花色的形成过程中起重要作用。依据七彩红竹转录组数据设计特异引物,采用ImPcR技术从七彩红竹中克隆获得了一个新的DFR基因cDNA全长,命名为IhDFR1（登录号为KF728205）。序列分析结果表明,IhDFR1基因cDNA全长945bp,编码314个氨基酸。生物信息学预测显示,该基因编码的蛋白具有典型的DFR蛋白功能结构域,存在2个特异结合位点,属于非Asn／Asp型DFR酶,与禾本科植物中的DFR具有较高的相似性。对不同发育时期七彩红竹的IhDFR1基因进行时空表达的结果显示,只有在竹秆颜色呈现红紫色时,IhDFR1基因才有表达。以上结果初步显示IhDFR1蛋白可能作为一个重要的酶参与竹秆花色素苷的代谢调控,同时为进一步研究七彩红竹花色素苷产生的分子机理和综合开发利用奠定了基础。     

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.     

水稻中大麦Mlo和玉米Hm1抗病基因同源序列的分析和定位

大麦抗病基因Mlo和玉米抗病基因Hm1编码的产物不具有绝大多数植物抗病基因产物所含有的保守结构域。这两个抗病基因的作用机理也不符合基因对基因学说。从水稻中分离克隆了Mlo基因的同源序列OsMlo-1和玉米Hm1基因的同源序列DFR－1。利用水稻分子标记遗传连锁图,将OsMlo-1定位于水稻第六染色体的两俱RZ667和RG424之间;Osmlo-1距离这两个分子标记分别为20．6和6．0cM(centi-Morgan)。将DFR－1定位于水稻第一染色体两个分子标记R2635和RG462之间;DFR－1距离这两个分子标记分别为11．3和23．9cM。参照已发表的水稻分子标记连锁图,发现OsMlo-1和DFR－1的染色体位点分别与两个报道的水稻抗稻瘟病数量性状位点（QTL）有较好的对应关系。结果提示,水稻中与大麦Mlo 和玉米Hml同源的基因可能也参于抗病反应的调控。     

苦荞二氢黄酮醇4-还原酶的原核表达与多克隆抗体制备

苦荞二氢黄酮醇4-还原酶(DFR)是花青素合成途径的关键酶。该研究以苦荞种子灌浆期cDNA为模板,采用RT-PCR方法克隆苦荞DFR编码基因,并将其连接到表达载体pET47b上,转化获得苦荞DFR编码基因的大肠杆菌BL21(DE3)工程菌,通过IPTG诱导表达,用SDS-PAGE分析表达产物,用亲和层析方法纯化蛋白,制备苦荞DFR多克隆抗体。RT-PCR技术获得了苦荞DFR编码基因的开放阅读框,重组表达载体经PCR和测序鉴定,表明表达载体构建成功,SDS-PAGE分析表达产物分别以可溶和不可溶的形式高效表达,亲和层析纯化得到融合蛋白,Western blotting显示,制备的多克隆抗体能特异识别其对应的抗原,天然的苦荞DFR蛋白在苦荞种子灌浆期中大量表达。苦荞DFR编码基因的原核表达与多克隆抗体的制备,为进一步开展DFR编码基因功能的研究奠定了基础。     

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。     

VIGS技术鉴定药用菊花皇菊中的DFR基因功能研究

从药用菊花皇菊中克隆二氢黄酮醇还原酶(dihydroflavonol 4-reductase, DFR)基因片段,插入病毒诱导载体中来抑制菊花内源性基因的表达,用于其基因功能分析。根据已报道的菊花DFR基因序列(GenBank登录号GU324979)设计引物,克隆部分基因序列,应用生物信息学方法对其氨基酸序列进行分析。采用病毒诱导基因沉默技术(VIGS),以菊花的扦插苗生长到第3～4片叶期的菊花植株为实验材料,沉默菊花的DFR基因,用半定量和荧光定量PCR检测病毒诱导沉默后DFR基因的表达。克隆得到黄菊DFR基因的最大开放阅读框为1 029 bp,共编码342个氨基酸,等电点是6.03,分子量是41 089.36,与同一科属植物之间同源性达到80%以上,说明DFR蛋白氨基酸序列在同一科属间具有高度保守性。根据DFR基因全长设计构建病毒诱导载体的引物得到453 bp的目的基因,命名为NbDFR。构建病毒载体(TRV)并利用携带目的基因的TRV重组载体病毒感染菊花幼苗,10 d后可以看出菊花出现发育迟缓且叶片皱缩干枯的现象,检测DFR基因发现基因表达下调约20%,但未达到显著性。DFR基因是否被成功沉默还进一步验证。病毒诱导的基因沉默技术可在药用菊花中初步快速鉴定相关基因的功能。     

日本蛇根草DFR3基因的克隆分析及其编码蛋白的分离纯化

二氢黄酮醇4-还原酶(DFR)作为花色苷代谢途径下游的关键酶,对植物花色苷的合成具有重要调控作用。该研究以日本蛇根草(Ophiorrhiza japonica)为材料,采用RT-PCR方法克隆获得一个DFR基因(OjDFR3),利用生物信息学方法对OjDFR3蛋白的性质进行了分析,通过实验完成该基因原核表达载体的构建及其重组蛋白的制备与纯化,为深入揭示日本蛇根草DFR基因的功能以及花色苷的合成与调控研究奠定基础。结果表明:(1)成功克隆获得一个DFR基因(OjDFR3);序列分析显示,OjDFR3基因cDNA全长为1 071 bp,可编码356个氨基酸,蛋白分子量为39.52 kD。(2)生物信息学分析表明,OjDFR3基因编码形成的蛋白由20种氨基酸组成,其中亮氨酸含量最多,不存在信号肽,是一种亲水性蛋白,定位在细胞质的可能性最大,三级结构由α螺旋、延伸链、无规则卷曲组成。(3)原核表达分析显示,重组质粒pET32a-OjDFR3可在大肠杆菌BL21(DE3)中表达,其最佳诱导表达条件为37℃、4 h、IPTG浓度为0.8 mmol/L,同时在100和200 mmol/L咪唑洗脱下的蛋白纯度最好。(4)按照上述最佳条件,制备并获得了大量浓度和纯度较好的蛋白。     

Complementation of the Tomato anthocyanin without (aw) Mutant Using the Dihydroflavonol 4-Reductase Gene

We isolated the dihydroflavonol 4-reductase (DFR) gene from tomato (Lycopersicon esculentum) using a previously characterized cDNA as probe. Earlier studies had indicated that the DFR gene is present in tomato as a single gene located on chromosome 2 near the locus anthocyanin without (aw). Mutant alleles of the aw locus result in the complete absence of anthocyanin pigmentation throughout all stages of plant development. When the genomic DFR clone was introduced by Agrobacterium-mediated transformation into plants bearing the aw mutation, primary transgenic seedlings accumulated anthocyanins that could be observed while the plants were still in tissue culture and which continued to be observed as the plants matured. Progeny of self pollinated and backcrossed transgenic plants segregated for anthocyanin pigmentation, and Southern hybridization analyses indicated the presence of the DFR transgene exclusively in those plants with pigmentation. These data indicate that the aw locus likely corresponds to the structural gene for DFR and that DFR can be used as a visual, nondestructive, plant-derived marker gene for tomato.     

Alteration of a single amino acid changes the substrate specificity of dihydroflavonol 4-reductase

Many plant species exhibit a reduced range of flower colors due to the lack of an essential gene or to the substrate specificity of a biosynthetic enzyme. Petunia does not produce orange flowers because dihydroflavonol 4-reductase (DFR) from this species, an enzyme involved in anthocyanin biosynthesis, inefficiently reduces dihydrokaempferol, the precursor to orange pelargonidin-type anthocyanins. The substrate specificity of DFR, however, has not been investigated at the molecular level. By analyzing chimeric DFRs of Petunia and Gerbera, we identified a region that determines the substrate specificity of DFR. Furthermore, by changing a single amino acid in this presumed substrate-binding region, we developed a DFR enzyme that preferentially reduces dihydrokaempferol. Our results imply that the substrate specificity of DFR can be altered by minor changes in DFR.     

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.     

The Rc and Rd genes are involved in proanthocyanidin synthesis in rice pericarp

Different colors, such as purple, brown, red and white, occur in the pericarp of rice. Here, two genes affecting proanthocyanidin synthesis in red- and brown-colored rice were elucidated. Genetic segregation analysis suggested that the Rd and A loci are identical, and both encode dihydroflavonol-4-reductase (DFR). The introduction of the DFR gene into an Rcrd mutant resulted in red-colored rice, which was brown in the original mutant, demonstrating that the Rd locus encodes the DFR protein. Accumulation of proanthocyanidins was observed in the transformants by the introduction of the Rd gene into the rice Rcrd line. Protein blot analysis showed that the DFR gene was translated in seeds with alternative translation initiation. A search for the Rc gene, which encodes a transacting regulatory factor, was conducted using available DNA markers and the Rice Genome Automated Annotation System program. Three candidate genes were identified and cloned from a rice RcRd line and subsequently introduced into a rice rcrd line. Brown-colored seeds were obtained from transgenic plants by the introduction of a gene containing the basic helix-loop-helix (bHLH) motif, demonstrating that the Rc gene encodes a bHLH protein. Comparison of the Rc locus among rice accessions showed that a 14-bp deletion occurred only in the rc locus.     

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.     

Cymbidium hybrida dihydroflavonol 4-reductase does not efficiently reduce dihydrokaempferol to produce orange pelargonidin-type anthocyanins

Some angiosperms are limited to a range of possible flower colors. This limitation can be due to the lack of an anthocyanin biosynthetic gene or to the substrate specificity of a key anthocyanin biosynthetic enzyme, dihydroflavonol 4-reductase (DFR). Cymbidium hybrida orchid flowers primarily produce cyanidin-type (pink to red) anthocyanins and lack the pelargonidin-type (orange to brick-red) anthocyanins. To investigate the underlying molecular mechanism of this flower color range, we cloned a Cymbidium DFR gene and transformed it into a DFR- petunia line. We found that the Cymbidium DFR did not efficiently reduce dihydrokaempferol (DHK), which is an essential step for pelargonidin production. Phylogenetic analysis of a number of DFR sequences indicate that the inability to catalyze DHK reduction has occurred at least twice during angiosperm evolution. Our results indicate that developing a pelargonidin-type orange flower color in Cymbidium may require the transformation of a DFR gene that can efficiently catalyze DHK reduction.