Molecular Cloning and Characterization of a DFR from Developing Seeds of Blue-grained Wheat in Anthocyanin Biosynthetic Pathway

Blue-grained wheat derived from the hybrid Triticum aestivum L. X Thinopyrum ponticum (Podp.) Barkworth et D. R. Dewey (Agropyron elongatum (Host) P. Beauv., 2n=70). The molecular biological mechanism of the biosynthetic pathway of blue pigments in the blue grain remains unclear yet. Dihydroflavonol 4-reductase (DFR) is one of the key enzymes controlling flavonoid synthesis in anthocyanin biosynthetic pathway, and may directly participate in the formation of blue pigment in the aleurone layer of blue-grained wheat. Here we cloned a DFR cDNA (TaDFR) from the developing seeds of blue-grained wheat, and four DFR genomic DNAs from Th. ponticum (ThpDFR.t), blue-grained wheat (TaDFR.bg), white-grained offspring of light blue-grained wheat (TaDFR.wg) and Chinese Spring (2n=42) (TaDFR.csg), respectively. TaDFR cDNA encodes a 354 amino-acids polypeptide with high identity to DFR from Hordeum vulgare L. (94%), Oryza sativa L. (83%), Zea mays L.(84%). The result of cluster analysis showed that TaDFR cDNA nucleotide sequence has 100% identity with that of TaDFR.csg. The four DFR genomic DNAs have extraordinary high homology and each has three introns. The differences of the four DFR genomic DNAs mainly exist in introns. Southern blotting analysis showed that there are at least 3-5 DFR copies in wheat, the copy numbers in different color grain wheats are not significantly different. The hybridization band patterns were the same, but different from that of Th. ponticum. DFR in blue-grained wheat belongs to a DFR superfamily. Northern blotting analysis indicated that the DFR expressed in the developing seeds of both blue- and white-grained wheat at 15 d after flowering (DAF), the mRNA levels of DFR reached the highest at 18 DAF, then declined quickly and disappeared at 33 DAF But the expression levels in blue-grained seeds were higher than that in white grain at the same seed developing stages. DFR transcripts accumulated in young leaves, and leaf sheaths of blue- and white-grained wheat and Th ponticum, but not detected in roots from different color wheats and developing seeds of Th. ponticum. Results indicated that there may exist some regulatory gene(s) which can increase the expression of DFR in the aleurone layer of blue-grained wheat, and thus resulting in the formation of blue pigments.

BnbHLH92a negatively regulates anthocyanin and proanthocyanidin biosynthesis in Brassica napus

Yellow seed trait is a desirable characteristic with potential for increasing seed quality and commercial value in rapeseed,and anthocyanin and proanthocyanidins(PAs)are major seed-coat pigments.Few transcription factors involved in the regulation of anthocyanin and PAs biosynthesis have been characterized in rapeseed.In this study,we identified a transcription factor gene BnbHLH92a(BnaA06T0441000ZS)in rapeseed.Overexpressing BnbHLH92a both in Arabidopsis and in rapeseed reduced levels of anthocyanin and PAs.Correspondingly,the expression profiles of anthocyanin and PA biosynthesis genes(TT3,BAN,TT8,TT18,and TTG1)were shown by quantitative real-time PCR to be inhibited in BnbHLH92a-overexpressing Arabidopsis seeds,indicating that BnbHLH92a represses the anthocyanin and PA biosynthesis pathway in Arabidopsis.BnbHLH92a physically interacts with the BnTTG1 protein and represses the biosynthesis of anthocyanins and PAs in rapeseed.BnbHLH92a also binds directly to the BnTT18 promoter and represses its expression.These results suggest that BnbHLH92a is a novel upstream regulator of flavonoid biosynthesis in B.napus.

Elucidation of the melitidin biosynthesis pathway in pummelo

Specialized plant metabolism is a rich resource of compounds for drug discovery.The acylated flavonoid glycoside melitidin is being developed as an anti-cholesterol statin drug candidate,but its biosynthetic route in plants has not yet been fully characterized.Here,we describe the gene discovery and functional characterization of a new flavonoid gene cluster(UDP-glucuronosyltransferases(Cg UGTs),1,2rhamnosyltransferase(Cg1,2Rha T),acyltransferases(Cg ATs))that is responsible for melitidin biosynthesis in pummelo(Citrus grandis(L.)Osbeck).Population variation analysis indicated that the tailoring of acyltransferases,specific for bitter substrates,mainly determine the natural abundance of melitidin.Moreover,3-hydroxy-3-methylglutaryl-Co A reductase enzyme inhibition assays showed that the product from this metabolic gene cluster,melitidin,may be an effective anti-cholesterol statin drug candidate.Co-expression of these clustered genes in Nicotiana benthamiana resulted in the formation of melitidin,demonstrating the potential for metabolic engineering of melitidin in a heterologous plant system.This study establishes a biosynthetic pathway for melitidin,which provides genetic resources for the breeding and genetic improvement of pummelo aimed at fortifying the content of biologically active metabolites.

钝裂银莲花花色素合成相关基因qRT-PCR内参基因的筛选

为了筛选适合于钝裂银莲花类黄酮/花青素合成途径中相关基因qRT-PCR表达分析时的内参基因,根据钝裂银莲花蓝/白不同花色花器官组织的转录组测序结果,选取了多聚泛素酶基因(polyubiquitin,UBQ)、微管蛋白基因(β-tubulin,β-TUB)、水通道蛋白基因(aquaporin,AQP)、肌动蛋白基因(actin,ACT)、甘油醛-3-磷酸-脱氢酶基因(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)、组蛋白基因(histone,HIS)、转录延伸因子基因(elongation factor 1-β,EF-1β)和60S核糖体蛋白基因(60S ribosomal protein L13-1,RPL13)等8个常用内参基因作为候选基因,以钝裂银莲花的叶片、茎杆和蓝/白色花器官等不同组织为试验材料,通过qRT-PCR检测这8个候选内参基因的表达情况,利用geNorm、NormFinder和BestKeeper等软件对其稳定性进行分析评价。结果表明:8个候选内参基因中,UBQ表现最稳定,而β-TUB相对稳定性最差。以最稳定的UBQ为内参对钝裂银莲花类黄酮/花青素合成途径中16个相关基因表达情况进行qRT-PCR分析,结果与前期转录组测序结果一致。UBQ为钝裂银莲花花色素合成途径相关基因表达分析的最适内参基因。

茶花红叶芽变品种‘金华美女’叶色突变相关主要化学成分含量变化

‘金华美女’是从‘贝拉大玫瑰’中选育出的茶花芽变品种,其主要特点是叶色呈现暗红色。分别测定了上述两个品种在4个不同发育阶段叶片叶绿素、类胡萝卜素、总花青苷、矢车菊素葡萄糖苷、总多酚、儿茶素和表儿茶素含量。结果表明:(1)在相同阶段,两个品种间叶绿素a和叶绿素b差异均不显著。认为红叶品种的叶绿素合成途径也正常,排除因叶绿素变异引起的叶色变异;(2)红叶品种‘金华美女’叶片中的类胡萝素含量显著低于绿叶品种‘贝拉大玫瑰’,可见其积累对‘金华美女’的叶色无影响;(3)在4个时期中,‘金华美女’叶片总多酚、儿茶素和表儿茶素的平均含量仅占‘贝拉大玫瑰’叶片中的相应含量的33.1%、42%和15%,可见变色品种叶片的多酚合成途径可能受到一定程度的阻碍。(4)‘金华美女’叶片中总花青苷平均含量达3.06 mg·g^(-1),是‘贝拉大玫瑰’的7倍。由此可见,叶片花青苷合成途径中红色类组分的积累和多酚合成途径中无色类组分的合成受阻,是引起‘金华美女’叶色变红的主要成因。

类黄酮合成途径结构基因的表达分析与中国水仙不能合成花青素原因的初步解析

植物类黄酮合成途径包括花青素、黄酮醇、原花青素等不同分支。为了解析中国水仙花色单一的原因,我们通过显色反应和HPLC分析了中国水仙(漳州水仙)不同器官中类黄酮的主要成分。结果显示:花瓣和副冠中类黄酮的主要成分是黄酮醇;鳞茎盘中的主要成分是原花青素;几个器官中都没有花青素。因此,缺乏花青素可能是中国水仙花色单一的主要原因。为了进一步了解中国水仙不能合成花青素的原因,我们进行了鳞茎盘转录组测序。De novo组装出36,006个unigene,平均读长706bp。通过Blast数据库比对、序列分析等方法鉴定类黄酮合成途径中表达的结构基因,共获得了4个Nt CHS、2个Nt CHI、3个Nt F3H、3个Nt UFGT、1个Nt F3’H、1个Nt DFR和1个Nt LAR;同时还获得了与类黄酮代谢相关的调控因子MYB,b HLH和WD40等基因。但没有发现花青素合成途径的ANS基因以及原花青素合成分支途径的ANR基因。通过q PCR研究获得的16个结构基因在中国水仙全开、半开和花蕾期三个时期的花瓣、副冠以及叶片和鳞茎盘中的表达。结果发现,在花瓣和副冠中Nt DFR的表达量低,Nt FLS的表达量很高;鳞茎盘中Nt DFR和Nt LAR的表达量都很高,Nt FLS的表达量低。结构基因的表达水平与这三种器官中类黄酮的主要成分相吻合。通过HPLC进一步分析了鳞茎盘中原花青素单体的成分,发现主要是儿茶素单体(catechin),说明中国水仙鳞茎盘中经过Nt DFR作用生成无色花青素(leucoanthocyandin)后,直接在Nt LAR的作用下合成原花青素,没有经过ANS和ANR的作用步骤,与转录组测序中没有发现ANS和ANR基因表达相一致。因此我们推测,中国水仙花瓣和副冠中也缺少ANS基因的表达,没有ANS基因的表达可能是中国水仙不能合成花青素的主要原因,有待进一步验证。

Phenylpropanoid Biosynthesis

The general phenylpropanoid metabolism generates an enormous array of secondary metabolites based on the few intermediates of the shikimate pathway as the core unit. The resulting hydroxycinnamic acids and esters are am- plified in several cascades by a combination of reductases, oxygenases, and transferases to result in an organ and devel- opmentally specific pattern of metabolites, characteristic for each plant species. During the last decade, methodology driven targeted and non-targeted approaches in several plant species have enabled the identification of the participating enzymes of this complex biosynthetic machinery, and revealed numerous genes, enzymes, and metabolites essential for regulation and compartmentation. Considerable success in structural and computational biology, combined with the an- alytical sensitivity to detect even trace compounds and smallest changes in the metabolite, transcript, or enzyme pattern, has facilitated progress towards a comprehensive view of the plant response to its biotic and abiotic environment. Trans- genic approaches have been used to reveal insights into an apparently redundant gene and enzyme pattern required for functional integrity and plasticity of the various phenylpropanoid biosynthetic pathways. Nevertheless, the function and impact of all members of a gene family remain to be completely established. This review aims to give an update on the various facets of the general phenylpropanoid pathway, which is not only restricted to common lignin or flavonoid biosynthesis, but feeds into a variety of other aromatic metabolites like coumarins, phenolic volatiles, or hydrolyzable tannins.