白菜型油菜DFR基因的克隆与序列分析

By using RACE (rapid amplification ofcDNA ends) based homologous cloning strategy, we have successfully isolated the genomic and full-length cDNA sequences of a gene encoding typical DFR (dihydroflavonol-4-reductase) from black-seeded Brassica campestris L. var. oleifera DC.. The gene, designated BcDFR here, is 1 722bp in length and harbors 5 introns with typical splice sites of plant DFR genes. BcDFR cDNA is 1311bp in length with a 1 158bp ORF as well as a 25bp 5‘ UTR and a 128bp 3‘ UTR. The encoded BcDFR protein is 385 aa with a calculated Mw of 42.85kD and a pI value of 5.55. The nucleotide and amino acid sequences of this gene share extensive homologies to plant DFR genes of wide origins especially high similarities to Cruciferous DFR genes. Sequence analyses such as phylogenetic analysis, conserved domain search and substrate specificity region detection all indicated that BcDFR gene is a quite potentially biofunctional gene. Its cloning enables us to further dissect the possible relatedness between DFR gene and Brassica seed coat color traits and to create transgenic novel yellow-seeded rapeseed germplasm through antisense- or RNAi-suppression of DFR gene expression in black-seeded elite cultivars.     

芥蓝二氢黄酮醇4–还原酶基因BoaDFR的克隆与功能鉴定

以白花芥蓝‘四季粗条’为材料克隆了Boa DFR基因,Gen Bank登录号为MT472647,CDS序列长为1158 bp,编码385个氨基酸,与甘蓝型油菜、甘蓝和白菜等具有高度同源性。Boa DFR表达水平在芥蓝不同发育时期、不同器官中存在显著差异,随植株发育总体呈现先下降后上升的趋势,在幼嫩种子中表达量最高。芥蓝原生质体的亚细胞定位结果显示Boa DFR定位在细胞核上。构建了农杆菌介导的芥蓝瞬时过表达体系,将Boa DFR转入白花芥蓝‘四季粗条’和黄花芥蓝‘福州黄花’中,发现Boa DFR在两种芥蓝中均高水平表达,转基因植株叶片颜色明显变紫,花青素苷显著积累,总含量最高达461.43μg·g-1 FW,而野生型和转空载体芥蓝叶片中几乎检测不到花青素苷。通过HPLC在转基因植株叶片中共检测到8种花青素苷,均为矢车菊花青素苷,其中矢车菊–3–阿魏酰–丙二酰–槐糖苷–5–葡萄糖苷占比最高。     

氮素对羽叶决明缩合单宁及相关酶活性的影响

缩合单宁对牧草品质的影响极大,适量的缩合单宁含量对于提高牧草的利用率和营养品质非常重要。研究以豆科牧草羽叶决明Cassia nictitoms为材料,分析了氮素梯度处理后茎和叶中缩合单宁的含量及其生物合成酶二氢黄酮醇还原酶(DFR)活性的变化特征。结果表明,羽叶决明叶片的缩合单宁含量高于茎,随氮素的增加,叶片中缩合单宁含量从14.78%下降到1.14%;茎中从4.62%开始降低,达到0.98%后趋于稳定;DFR活性表现出与缩合单宁相似的变化规律,二者在叶和茎中都呈显著(P0.01)的二次曲线关系。植株粗蛋白含量测定发现,随氮素的增加粗蛋白含量无显著增加(P0.05)。因此,适当增加氮素的使用量可以达到减少缩合单宁含量,从而提高羽叶决明的利用率和营养品质。     

荔枝DFR基因的克隆及其序列分析

采用同源克隆的方法从荔枝果皮中克隆得到一个二氢黄酮醇4-还原酶（DFR）基因,该基因开放阅读框全长1062bp,编码353个氨基酸．基因组扩增得到长度为2321 bp的片段,分析发现：该基因含有5个内含子,分别在119-255、426-1158、1353-1470、1632-1819、2013-2095bp之间．通过系统发育分析发现,该基因编码的蛋白与山葡萄、海棠和山竹子等果树具有较近的亲缘关系．     

滇牡丹(Paeonia delavayi)二氢黄酮醇4-还原酶基因的鉴定及表达

为了研究二氢黄酮醇4-还原酶基因(DFR)对滇牡丹(Paeonia delavayi)花瓣中花色形成的作用,本研究采用RT-PCR技术首次从滇牡丹中克隆得到一个具有完整开放阅读框的二氢黄酮醇4-还原酶基因(PdDFR),生物信息学分析和转录模式分析显示,该基因全长1 050 bp,共编码349个氨基酸,其蛋白序列与黄牡丹(Paeonia lutea) DFR的蛋白序列具有99.00%的一致性。序列比对分析显示PdDFR基因存在一个由21个氨基酸组成的NADPH结合位点"VTGATGYIGSWLVKTLLERGN"。滇牡丹PdDFR蛋白与黄牡丹(Paeonia lutea, ALX35996.1)、蓖麻(Ricinus communis, XP015577076.1)、木薯(Manihot esculenta, XP021607-041.1)的DFR蛋白聚为一类;转录模式分析表明PdDFR基因在红色花瓣中有表达,在根、茎、叶中不表达。本研究通过对滇牡丹转录组数据的分析,分离并克隆得到PdDFR基因,为利用基因工程技术选育优良滇牡丹花卉品种提供理论参考。     

石榴花二氢黄酮醇还原酶（DFR）基因RT—PCR扩增体系的建立与优化

分别以红花和粉花石榴‘泰山红’、‘重瓣粉花’盛花期花瓣为材料，提取RNA后进行反转录，对DFR基因的PCR扩增体系进行了优化，建立了高效、特异的石榴花DFR基因的RT—PCR扩增体系，为克隆基因全长奠定了基础。     

Breeding of transgenic orangePetunia hybrida varieties

Summary Flower colour is the major contributor to the total ornamental value of a flower. The combination of biochemical knowledge and genetic engineering technology has resulted in the addition of a new colour to the existing colour range ofPetunia hybrida. This has been achieved by expression of the maize dihydroflavonol-4-reductase (DFR) gene in a suitable petunia acceptor which leads to the accumulation of pelargonidin-derived pigments in flowers. The resulting flower colour, however, was a pale brick-red, which is commercially unattractive in petunia. Our objective was to produce a product suitable for commercialisation by introducing the DFR gene into our breeding material via normal sexual recombination. Although the initial transformant exhibited many negative characteristics, first analyses indicated that it was feasible to obtain material for creating commercial hybrids. Experimental hybrids based on F₄ lines were obtained with improved phenotypical expression of the orange flower colour in combination with a good general performance. In order to assess consumer-related characteristics, selected experimental hybrids were tested under field conditions. All transgenic plants had a normal appearance when compared to non-transgenic control plants. No linkage was observed between the transgenic trait and any negative characteristic. From these studies it can be concluded that through a combination of biochemistry, breeding and genetic engineering it is possible to generate unique flower colours in a cultivar with commercial potential.     

石榴等观赏植物DFR基因生物信息学分析

根据GenBank中已登录的红花石榴、粉花石榴、姜荷花、芍药、水母雪莲、大丽花、瓜叶菊和兰花等植物二氢黄酮醇4-还原酶(DFR)基因的核苷酸序列和氨基酸序列,应用生物信息学软件对其核苷酸序列的外显子、氨基酸序列的理化性质、疏水性/亲水性和跨膜结构等进行了预测。结果表明,这几种植物DFR基因均存在1个外显子,含量最丰富的氨基酸是Ala、Gly、Cys和Thr;除红花石榴和粉花石榴DFR属于不稳定蛋白外,其余植物均属于稳定蛋白。这几种植物DFR蛋白均为亲水性蛋白,存在明显的疏水区和亲水区以及跨膜结构等。     

甜樱桃DFR基因内含子2和内含子3的多态性

【目的】研究70个甜樱桃品种DFR基因多态性与果皮颜色的相关性。【方法】通过DNA序列分析,以不同果皮颜色的10个甜樱桃品种（Prunus avium L.）为材料,检测DFR基因的多态性。根据多态性出现的位点设计特异引物,通过PCR扩增检测70个甜樱桃品种DFR基因的多态性。【结果】获得甜樱桃DFR基因约1 kb的片段,测序结果用BLAST分析发现,其核苷酸序列与樱桃李（Prunus cerasifera）的核苷酸相似性为80 %,预测的氨基酸序列与已知的甜樱桃（P. avium）DFR氨基酸序列相似性达99 %。该片段由3个外显子和3个内含子组成,2个多态性位点分别在内含子2和内含子3上。在黄色、黄底红晕和红色果皮3个组的70个甜樱桃品种中发现3个单倍型,共5种单倍型组合。通过SAS 9.0软件分析发现,所检测到的DFR基因的等位基因频率在黄底红晕果皮品种组和红色品种果皮组中的分布无显著差异。【结论】本研究在甜樱桃DFR基因座上得到2个差异位点,分别在内含子2和内含子3内,其优势基因频率依次为：0.864和0.679;但未发现DFR基因内含子2和内含子3的多态性与果皮颜色之间存在直接关系。     

芸薹属DFR基因家族RNA干扰载体的构建

二氢黄酮醇-4-还原酶(dihydroflavonol-4-reductase,DFR)是类黄酮途径的一个关键酶,对种皮色泽和花色的形成具有重要作用。本研究从甘蓝型油菜克隆了600 bp(不计人工酶切位点)的芸薹属DFR基因家族的RNA干扰片段BDFRI,将其反义片段和正义片段分别插入到本课题组新近改造的植物RNAi平台载体pFGC5941M的启动子与间隔区之间、间隔区与终止子之间,构建了11 400 bp的RNAi干扰载体pFGC5941M-BDFRI,通过多重PCR鉴定证实载体构建成功,并转化到根癌农杆菌菌株LBA4404中形成了工程菌株。此载体的构建为进一步研究DFR基因参与决定芸薹属种皮色素和茎叶彩色性状的分子机理和代谢调控奠定了基础。