大豆籽粒发育过程中异黄酮合成相关酶基因的表达模式与异黄酮积累的相关分析

利用高效液相色谱法和实时定量PCR方法,分别测定了2个异黄酮含量显著差异的大豆品种鲁黑豆2号(LHD2)和南汇早黑豆(NHZ)在子粒发育过程中的异黄酮含量变化以及异黄酮合成相关酶基因的表达模式变化,试图分析异黄酮积累与各基因表达量变化的相关关系。结果表明在大豆子粒发育过程中,异黄酮含量逐渐升高,而不同异黄酮合成相关酶基因的表达趋势不同,CHS7、CHS8、CHR、CHI1A和IFS2的表达趋势与异黄酮积累模式基本一致,而IFS1和CHI1B1的表达趋势与异黄酮积累模式相反。IFR的表达模式在2个大豆品种中存在相反的趋势,在LHD2中与异黄酮组分积累趋势相反,而在NHZ中与异黄酮组分积累趋势相同。结果还表明,同一基因家族中不同基因在子粒发育过程中的表达量也存在差异。查尔酮合酶基因家族中CHS7和CHS8以及查尔酮异构酶基因家族的CHI1A的表达水平相对其他成员较高,异黄酮合酶基因家族中IFS2的表达量显著高于IFS1的表达量,预示这些基因家族在大豆子粒异黄酮积累过程中存在功能分化。此外,各基因表达模式与异黄酮积累的相关分析结果表明,不同基因表达模式与异黄酮积累的相关性在2个品种中也不尽相同。LHD2中CHS7、CHS8和IFS2在子粒发育过程中的表达量变化与不同异黄酮组分呈显著正相关,CHI1B1基因的表达量变化与不同异黄酮组分呈显著负相关。而在NHZ中,IFR在子粒发育过程中的表达量变化与多个异黄酮组分呈显著正相关。这预示了不同大豆品种异黄酮含量差异的潜在遗传基础。各异黄酮合成相关酶基因表达量变化的相关分析表明,在2个品种中,苯丙氨酸水解酶PAL1与4CL,4CL与CHS2以及CHS1与IFS2基因的表达量均呈现显著正相关。表明这些基因可能通过协同作用共同调控异黄酮的合成与积累。这些结果为今后利用基因工程提高大豆异黄酮含量奠定了基础。     

花青素生物合成关键酶的研究进展

花青素是植物花呈现不同色彩的物质基础,其生物合成途径主要受到查尔酮合成酶(CHS)、查尔酮异构酶(CHI)、黄烷酮3-羟化酶(F3H)、类黄酮3'-羟化酶(F3'H)、类黄酮3’,5’-羟化酶(F3'5'H)、二羟基黄酮醇还原酶(DFR)、花色素苷合成酶(ANS)以及类黄酮3-O-糖基转移酶(UFGT)等关键酶的控制.主要介绍花青苷生物合成途径、关键酶晶体结构及利用基因工程改造花色的研究进展,讨论目前花色改造存在的问题,并对今后的研究前景进行展望.     

大豆异黄酮合成途径相关基因差异表达分析

大豆异黄酮是一种应用广泛、具有医用和保健功能的活性物质。为揭示异黄酮合成途径相关基因表达差异,本研究采用实时定量PCR技术分析相关基因在不同大豆品种、发育时期及组织部位的表达。结果发现,苯丙氨酸解氨酶基因PAL、肉桂酸羟化酶基因C4H、香豆酸辅酶A连接酶基因4CL在高异黄酮品种中豆27 R2期叶片中的表达量显著高于低异黄酮品种楚秀;查尔酮合成酶基因CHS、异黄酮合成酶基因IFS在中豆27 R8期子粒中的表达量显著高于楚秀;细胞色素还原酶基因CPR在中豆27 R7期叶片与子粒的表达量与楚秀相比显著降低。这些差异表达的基因可能是形成大豆品种异黄酮含量高低的重要原因。     

定点突变提高虎杖聚酮合酶的苯亚甲基丙酮合酶活性北大核心CSCD

虎杖(Polygonum cuspidatum)聚酮合酶(polyketide synthase 1,PcPKS1)同时具有查尔酮合酶(chalcone synthase,CHS)及苯亚甲基丙酮合酶(benzylidene acetone synthase,BAS)催化活性,能够催化生成聚酮类化合物柚皮素查尔酮和苯亚甲基丙酮,进而催化合成黄酮类或覆盆子酮等具有多种生物学活性的化合物。本研究通过分析虎杖PcPKS1与掌叶大黄(Rheum palmatum)BAS、拟南芥(Arabidopsis thaliana)CHS等家族成员的序列以及酶催化位点的构象,确定可能影响酶功能的3个氨基酸位点:Thr133、Ser134、Ser339。采用定点突变对PcPKS1进行分子修饰,成功获得2个突变体并进行相关体外酶促反应,高效液相色谱(high performance liquid chromatography,HPLC)产物分析结果表明,在pH 7.0和pH 9.0的体外酶促条件下,突变体T133LS134A和S339V维持BAS和CHS双功能活性,且BAS活性显著高于原PcPKS1。本研究为利用PcPKS1进行基因工程调节黄酮类和覆盆子酮化合物的生物合成提供理论依据。     

非洲菊查尔酮合酶基因的克隆、序列分析及在大肠杆菌中的表达

利用RT-PCR方法,从非洲菊(Gerbera hybrida)花瓣的CDNA中克隆到了查尔酮合酶(Chalcone Synthase,CHS)基因CHS,进行了序列分析。结果表明,克隆到的CHS基因全长为1197bps,编码一个由398个氨基酸残基组成的多肽,与Helariutta等发表的非洲菊查尔酮合酶CHSI基因的CDNA序列的CHS基因同源性高达99％。进一步将该基因克隆到表达载体pET32a上,经IPTG诱导表达,得到高效表达的融合蛋白。     

大豆异黄酮合酶基因的克隆及序列分析

目的:大豆异黄酮是多酚类混合物,有防治肿瘤发生,提高机体免疫力等多种保健功能。异黄酮合酶(isoflavone synthase,IFS)是合成异黄酮的关键酶。本文为了利用异黄酮的特有生物学功能,从大豆中克隆了该基因。方法:采用PCR扩增从大豆[Glycine max(Linn.)Merr.]总RNA中分离了异黄酮合酶基因,并将其克隆到pUCm-T载体并测序。结果:得到全长1583bp的片段。以期用于构建诱导表达基因敲除系统,并用于无性繁殖植物的无标记基因转化。结论:序列分析表明,异黄酮合酶基因(IFS1)含1583个核苷酸,与已报道的序列比较,核苷酸的同源性为92%。     

一个新的甘薯查尔酮异构酶基因的克隆和表达分析

查尔酮异构酶(chalcone isomerase,CHI)是花青素生物合成途径中的一个关键酶。本研究根据转录组数据,利用RT-PCR技术在甘薯中克隆了一个新的CHI基因,命名为Ib CHIL1。Ib CHIL1基因c DNA长857 bp,包含1个621 bp的开放阅读框,编码206个氨基酸。聚类分析表明,Ib CHIL1属于Ⅳ型CHI成员,和其他植物中的类似蛋白具有很高的同源性。表达分析显示,Ib CHIL1主要在紫肉甘薯中表达,与花青素积累正相关,推测该基因在甘薯花青素生物合成过程中具有重要的生物学功能。     

石榴果色合成相关基因CHS和CHI的表达特性分析

花色苷是类黄酮家族中重要的一类次生代谢产物,对果实呈色起重要作用。CHS (查尔酮合成酶)和CHI (查尔酮异构酶)为花色苷合成提供了前体物质,是花色苷合成所不可或缺的。利用RT-PCR和RACE方法,本研究从石榴果皮中克隆了与花色苷合成相关的CHS基因和CHI基因的cDNA全长,同时采用qRT-PCR研究了这两个基因在三个不同色泽石榴品种‘红宝石’、‘水晶甜’、‘墨石榴’发育期内的表达模式,并分析了果皮花色苷含量变化与基因转录水平的关系。结果表明,石榴中CHS和CHI基因cDNA全长分别为1 197 bp和693 bp,分别编码398和230个氨基酸,命名为PgCHS和PgCHI,在GenBank中的登录号分别为KF841615和KF841616。在氨基酸水平上,Pg CHS与荔枝、葡萄、山竹等果树的同源性达到90%以上。Pg CHI与果树中龙眼、梨、美洲葡萄、桑树等同源性达到70%以上。qRT-PCR结果显示,CHS和CHI基因的表达模式随色泽发育期和品种不同而有差异。在‘红宝石’石榴中,该两个基因都有前期和后期两个表达高峰期;而‘水晶甜’石榴中这两个基因的表达高峰期均出现在中后期;‘墨石榴’发育初期时CHS和CHI的表达量最高,以后的表达量都较低。同一品种内,CHS和CHI的表达具有协同性,两者的协同性表达有利于花色苷及其他类黄酮相关产物的合成。3个品种中CHS和CHI基因的表达与花色苷的积累并不一致。     

虎杖苯亚甲基丙酮合酶PcPKS2的表达纯化和晶体生长

植物类型Ⅲ聚酮合酶超家族(PKSs),又称查尔酮合酶(Chalcone synthase,CHS)超家族,催化合成多种植物次生代谢产物的分子骨架。苯亚甲基丙酮合酶(Benzalacetone synthase,BAS)催化4-香豆酰辅酶A与丙二酰辅酶A通过一步脱羧缩合反应生成苯亚甲基丙酮,是一系列具有重要生物学活性苯丁烷类化合物及其衍生物的前体化合物。前期工作从虎杖中分离出苯亚甲基丙酮合酶BAS(PcPKS2)和1个具有CHS和BAS活性的双功能酶(PcPKS1)。两者与超家族其他成员序列经比较,在包括门卫氨基酸Phe215和Phe265在内的重要氨基酸序列存在一定差异。已有蛋白晶体学研究结果表明,PKSs家族不同成员的功能多样性来自于酶催化位点的非常微小的构象变化。为了能够从结构上比较PcPKS2和Pc PKS1双功能酶活性差异可能产生的机制,以确定其高效BAS活性的分子机理,研究利用了大肠杆菌原核表达系统过量表达了C-端融合有His6标签的重组蛋白,经纯化得到了高纯度蛋白。经过对其晶体生长条件进行摸索和优化,得到了能用于X-射线衍射的单晶,为其结构解析、催化机理研究、了解虎杖聚酮类化合物生物合成机制和该类酶在基因工程中的应用提供了基础。     

查尔酮合酶基因对转基因植物花色和育性的影响

查尔酮合酶 ( chalcone synthase,CHS)是花色素合成途径中的一个关键酶 ,它在植物中表达量的改变可能影响花的颜色。从矮牵牛 ( Petunia hybrida)特定发育阶段的花瓣的 c DNA中 ,克隆到查尔酮合酶基因 ,并正向插入到原核表达载体和含有花椰菜花叶病毒 Ca MV 35 S启动子的真核表达载体中 ,在原核中得到高效表达 ,并通过土壤农杆菌介导的方法转化矮牵牛。转基因植物的花色不但发生了明显的变异 ,其育性也受到了影响 ,不能产生正常花粉粒 ,成为雄性不育植株。 Northern杂交表明 ,转基因植物花瓣中 ,内源及外源查尔酮合酶基因转录均受到抑制     

Polymorphism and expression of isoflavone synthase genes from soybean cultivars

Isoflavones are synthesized by isoflavone synthases via the phenylpropanoid pathway in legumes. We have cloned two isoflavone synthase genes, IFS1 and IFS2, from a total of 18 soybean cultivars. The amino acid residues of the proteins that differed between cultivars were dispersed over the entire coding region. However, amino acid sequence variation did not occur in conserved domains such as the ERR triad region, except that one conserved amino acid was changed in the IFS2 protein of the GS12 cultivar (R374G) and the IFS1 proteins of the 99M06 and Soja99s65 cultivars (A109T, F105I). In three cultivars (99M06, 99M116, and Simheukpi), most of amino acid changes were such that the difference between the amino acid sequences of IFS1 and IFS2 was reduced. The expression profiles of three enzymes that convert naringenin to the isoflavone, genistein, chalcone isomerase (CHI), isoflavone synthase (IFS) and flavanone 3-hydroxylase (F3H) were examined. In general, IFS mRNA was more abundant in etiolated seedlings than mature plants whereas the levels of CHI and F3H mRNAs were similar in the two stages. During seed development, IFS was expressed a little later than CHI and F3H but expression of these three genes was barely detectable, if at all, during later seed hardening. In addition, we found that the levels of CHI, F3H, and IFS mRNAs were under circadian control. We also showed that IFS was induced by wounding and by application of methyl jasmonate to etiolated soybean seedlings.     

Multigene synergism increases the isoflavone and proanthocyanidin contents of Medicago truncatula

Isoflavones and proanthocyanidins (PAs), which are flavonoid derivatives, possess many health benefits and play important roles in forage‐based livestock production. However, the foliage of Medicago species accumulates limited levels of both isoflavones and PAs. In this study, biosynthesis of isoflavone and PA in Medicago truncatula was enhanced via synergy between soya bean isoflavone synthase (IFS1); two upstream enzymes, chalcone synthase (CHS) and chalcone isomerase (CHI); and the endogenous flavanone 3‐hydroxylase (F3H). Constitutive expression of GmIFS1 alone resulted in ectopic accumulation of the isoflavone daidzein and large increases in the levels of the isoflavones formononetin, genistein and biochanin A in the leaves. Furthermore, coexpression of GmIFS1 with GmCHS7 and GmCHI1A generally increased the available flux to flavonoid biosynthesis and resulted in elevated isoflavone, flavone and PA contents. In addition, down‐regulation of MtF3H combined with coexpression of GmIFS1, GmCHS7 and GmCHI1A led to the highest isoflavone levels (up to 2 μmol/g fresh weight in total). Taken together, our results demonstrate that multigene synergism is a powerful means to enhance the biosynthesis of particular flavonoids and can be more broadly applied to the metabolic engineering of forage species.     

Modification of phenolic metabolism in soybean hairy roots through down regulation of chalcone synthase or isoflavone synthase

Soybean hairy roots, transformed with the soybean chalcone synthase (CHS6) or isoflavone synthase (IFS2) genes, with dramatically decreased capacity to synthesize isoflavones were produced to determine what effects these changes would have on susceptibility to a fungal pathogen. The isoflavone and coumestrol concentrations were decreased by about 90% in most lines apparently due to gene silencing. The IFS2 transformed lines had very low IFS enzyme activity in microsomal fractions as measured by the conversion of naringenin to genistein. The CHS6 lines with decreased isoflavone concentrations had 5 to 20-fold lower CHS enzyme activities than the appropriate controls. Both IFS2 and CHS transformed lines accumulated higher concentrations of both soluble and cell wall bound phenolic acids compared to controls with higher levels found in the CHS6 lines indicating alterations in the lignin biosynthetic branch of the pathway. Induction of the soybean phytoalexin glyceollin, of which the precursor is the isoflavone daidzein, by the fungal pathogen Fusarium solani f. sp. glycines (FSG) that causes soybean sudden death syndrome (SDS) showed that the low isoflavone transformed lines did not accumulate glyceollin while the control lines did. The (iso)liquritigenin content increased upon FSG induction in the IFS2 transformed roots indicating that the pathway reactions before this point can control isoflavonoid synthesis. The lowest fungal growth rate on hairy roots was found on the FSG partially resistant control roots followed by the SDS sensitive control roots and the low isoflavone transformants. The results indicate the importance of phytoalexin synthesis in root resistance to the pathogen. Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

A genomic approach to isoflavone biosynthesis in kudzu (<Emphasis Type="Italic">Pueraria lobata</Emphasis>)

Roots of kudzu (Pueraria lobata) are a rich source of isoflavone O- and C-glycosides. Although O-glycosylation of (iso)flavonoids has been well characterized at the molecular level, no plant isoflavonoid C-glycosyltransferase genes have yet been isolated. To address the biosynthesis of kudzu isoflavonoids, we generated 6,365 high-quality expressed sequence tags (ESTs) from a subtraction cDNA library constructed using RNA from roots that differentially accumulate puerarin. The ESTs were clustered into 722 TCs and 3,913 singletons, from which 15 family I glycosyltransferases (UGTs) were identified. Hierarchical clustering analysis of the expression patterns of these UGTs with isoflavone synthase (IFS) in a range of tissues identified UGTs with potential functions in isoflavone glycosylation. The open reading frames of these UGTs were expressed in E. coli for functional analysis, and one was shown to preferentially glycosylate isoflavones at the 7-O-position. In addition, ESTs corresponding to chalcone synthase, chalcone reductase, chalcone isomerase (CHI) and 2-hydroxyisoflavanone dehydratase were identified. Recombinant CHI proteins had high activities with both 6′-deoxy- and 6′-hydroxy chalcones, typical of Type II CHIs. Establishment of this EST database and identification of genes associated with kudzu isoflavone biosynthesis and glycosylation provide a new resource for metabolic engineering of bioactive kudzu isoflavones.     

Partial reconstruction of flavonoid and isoflavonoid biosynthesis in yeast using soybean type I and type II chalcone isomerases

Flavonoids and isoflavonoids are major plant secondary metabolites that mediate diverse biological functions and exert significant ecological impacts. These compounds play important roles in many essential physiological processes. In addition, flavonoids and isoflavonoids have direct but complex effects on human health, ranging from reducing cholesterol levels and preventing certain cancers to improving women's health. In this study, we cloned and functionally characterized five soybean (Glycine max) chalcone isomerases (CHIs), key enzymes in the phenylpropanoid pathway that produces flavonoids and isoflavonoids. Gene expression and kinetics analysis suggest that the soybean type I CHI, which uses naringenin chalcone as substrate, is coordinately regulated with other flavonoid-specific genes, while the type II CHIs, which use a variety of chalcone substrates, are coordinately regulated with an isoflavonoid-specific gene and specifically activated by nodulation signals. Furthermore, we found that some of the newly identified soybean CHIs do not require the 4′-hydroxy moiety on the substrate for high enzyme activity. We then engineered yeast (Saccharomyces cerevisiae) to produce flavonoid and isoflavonoid compounds. When one of the type II CHIs was coexpressed with an isoflavone synthase, the enzyme catalyzing the first committed step of isoflavonoid biosynthesis, various chalcone substrates added to the culture media were converted to an assortment of isoflavanones and isoflavones. We also reconstructed the flavonoid pathway by coexpressing CHI with either flavanone 3β-hydroxylase or flavone synthase II. The in vivo reconstruction of the flavonoid and isoflavonoid pathways in yeast provides a unique platform to study enzyme interactions and metabolic flux.     

Design of tandem genes cluster and expression vector for biosynthesis of soybean isoflavones

A tandem gene cluster CHS-CHI-IFS (rIFS) for secondary metabolites of plant isoflavones was constructed by using the chalcone synthase (CHS), chalcone isomerase (CHI), and isoflavone synthase (IFS) (GenBank accession numbers {"type":"entrez-nucleotide","attrs":{"text":"EU526827","term_id":"170783761","term_text":"EU526827"}}EU526827, {"type":"entrez-nucleotide","attrs":{"text":"EU526829","term_id":"170783765","term_text":"EU526829"}}EU526829, {"type":"entrez-nucleotide","attrs":{"text":"EU526830","term_id":"170783767","term_text":"EU526830"}}EU526830) in a single recombination event with the pET22b vector. The resulting expression vector pET-rIFS was heterogeneously expressed. The highlights of the vector include ease of handling, high efficiency and universal application among diverse plant species. To the best of our knowledge, this is the first attempt at developing a novel method of constructing tandem gene cluster for future research involving secondary metabolism of isoflavones and isoflavones engineering.Key words: Isoflavones biosynthesis, Novel method, Secondary metabolism, Tandem gene cluster     

Characterization and expression analysis of chalcone synthase and chalcone isomerase genes in Phyllanthus emblica (L.)

Journal of Plant Biochemistry and Biotechnology - The two decisive enzymes in flavonoid biosynthetic pathway are chalcone synthase (CHS) and chalcone isomerase (CHI), wherein the former carries the...