植物查尔酮异构酶研究进展

黄酮类化合物属于多酚类次生代谢物,具有广泛的药用价值。查尔酮异构酶（CHI）是黄酮类代谢途径中的一个关键酶,催化分子内环化反应,使双环的查尔酮转化为有生物学活性的三环（2S）-黄烷酮。植物体内的CHI活性与类黄酮物质的合成有着密切联系,CHI转基因研究对于提高植物类黄酮含量有重要意义。简要概述了查尔酮异构酶的结构特点、催化反应机理以及CHI转基因的研究进展。     

查尔酮合酶基因的克隆、全序列分析及在大肠杆菌中的高效表达

类黄酮是植物中的一种重要的次级代谢产物,它与植物的花色形成有关。查尔酮合酶(Chalcone synthase,CHS)是类黄酮合成途径中的一个关键酶,在植物体内,CHS表达量的增加或减少都可能改变花的颜色。从矮牵牛(Petunia hybrida)花瓣的cDNA中克隆到了CHS—A基因,进行了全序列分析,并与国外已报道的CHS—A序列进行了同源性比较。结果表明,克隆的CHS-A基因长为1170bp,编码一个由389个氨基酸组成的多肽,与国外已报道的CHS—A同源率高达99％。此外,还在大肠杆菌中实现了CHS—A基因的高效表达。CHS—A基因的成功克隆与表达为研究CHS—A基因对植物花色的影响打下了一个良好的基础。     

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.     

查尔酮合酶基因的克隆、全序列分析及在大肠杆菌中的高效表达

类黄酮是植物中的一种重要的次级代谢产物,它与植物的花色形成有关。查尔酮合酶是类黄酮合成途径中的一个关键酶,在植物体内,ＣＨＳ表达量的增加或减少都可能改变花的。从矮牵牛花瓣的ｃＤＮＡ中克隆到了ＣＨＳ－Ａ基因,进行了全序列分析,并与国外已报道的ＣＨＳ－Ａ－序列进行了同源性比较。     

罗汉果查尔酮合成酶基因的生物信息学分析

查尔酮合成酶(chalcone synthase,CHS)是类黄酮生物合成的关键酶,在植物发育、防止UV损伤、抗病和逆境反应中起着重要作用。本研究通过EST测序,获得了罗汉果查尔酮合成酶基因序列(登录号:GU980155)。为了进一步了解罗汉果查尔酮合成酶基因的特征,我们将其与46种植物的查尔酮合成酶基因的核酸序列和氨基酸序列进行比对和进化分析。结果表明,罗汉果查尔酮合成酶基因的核酸序列和氨基酸序列与其它物种的查尔酮合成酶基因均具较高同源性,编码区相似性约为94%。使用PHYLIP和MEGA4分别构建了邻接树、最大似然树和最大简约树,但经bootstrap检验,最优树未能明确罗汉果查尔酮合成酶基因的系统发育地位。以紫花苜蓿查尔酮合成酶的三维结构为参考,利用同源建模的方法预测了罗汉果查尔酮合成酶的三维结构,发现罗汉果查尔酮合成酶具有保守的活性位点和空间结构。     

水母雪莲查尔酮合酶基因的克隆、表达及酶活分析

从水母雪莲Saussurea medusa Maxim. cDNA文库中得到一段查尔酮合酶基因 (SmCHS) 片段,然后通过RT-PCR得到完整的查尔酮合酶基因cDNA。序列分析表明SmCHS全长1 313 bp,其开放阅读框为1 170 bp,编码389个氨基酸,预测表达蛋白的分子量为43 kDa。构建原核表达质粒pET28a(+)-SmCHS,重组质粒转化大肠杆菌BL21(DE3),获得表达菌株。经IPTG诱导表达后,对表达产物进行SDS-PAGE分析,结果显示,表达的融合蛋白以部分可溶的形式存在。用Ni-NTA预装柱对融合蛋白进行亲和纯化,对纯化蛋白进行酶活检测,结果表明融合蛋白具有查尔酮合酶活性,可催化底物4-香豆酰辅酶A和丙二酰辅酶A缩合生成产物柚皮素查尔酮。     

芸蔓属查尔酮异构酶基因家族RNA干扰载体的构建

作为类黄酮途径的第二步关键酶,查尔酮异构酶(chalcone isomerase,CHI)催化四轻基查尔酮成为抽皮素,用于合成各种类黄酮物质,影响多种性状.芸蔓属含有多种重要的油料、蔬菜和观赏植物.本研究从甘蓝型油菜克隆了芸蔓属CHI基因家族的干扰片段BCHII(761 bp),采用NcoI+AatII和BarnHI+XbaI双酶切方案分别将其反义片段和正义片段亚克隆到植物RNA干扰(RNAi)平台载体pFGC5941M的启动子与间隔区、间隔区与终止子之间,形成了11674by的RNAi载体pFGC5941M-BCHII(简称pBCHID,多种PCR检测表明2个片段的插入方向正确,重组载体完整,将其转化根癌农杆菌菌株LBA4404以后获得了工程菌株LBA4404-pBCHII-.本研究结果将促进研究CHI与芸蔓属异花授粉、植株色彩、黄籽和抗逆性等性状的关系和开展相关分子育种.     

低温促进毛竹叶片类黄酮合成及相关基因的表达模式分析

类黄酮在植物耐低温胁迫方面发挥着重要作用,为揭示低温对毛竹(Phyllostachys edulis)叶片中类黄酮合成的影响,采用分光光度法测定了不同生长时期和低温胁迫下毛竹幼苗叶片中的类黄酮含量,通过生物信息学方法对毛竹类黄酮早期生物合成关键酶基因进行了鉴定,并用q PCR方法分析了其表达模式。结果表明,随着叶片的生长,类黄酮含量呈现先升高后降低的趋势,而低温下,功能叶片中类黄酮含量则呈现上调趋势,且在8 h时达极显著水平。在毛竹基因组中鉴定了类黄酮早期生物合成3个酶基因家族共29个成员,包括20个查尔酮合酶基因(Pe CHSs)、8个查尔酮异构酶基因(Pe CHIs)和1个黄酮-3-烃化酶基因(Pe F3H1),这些基因的启动子中均含有响应低温及其他非生物胁迫的调控元件。Pe CHSs倾向在根和叶中表达,而PeCHIs为组成型表达。在不同生长时期的叶片中,仅PeCHS1表达与类黄酮的含量变化趋势一致;而低温胁迫下,3个PeCHSs、2个PeCHIs和PeF3H1在功能叶片中呈上调表达,与类黄酮含量变化趋势一致。因此,毛竹可能通过提高类黄酮早期生物合成酶基因的表达量促进类黄酮的合成来...     

干旱胁迫及复水对马铃薯类黄酮合成途径中关键酶及基因表达的影响

为研究干旱胁迫及复水对马铃薯(Solanum tuberosum)类黄酮合成途径影响的机制,以‘克新1号’为试验材料,利用高通量测序技术分析了苗期干旱处理(40%土壤相对含水量维持14 d,DT)与对照(70%土壤相对含水量维持14 d,DCK)、复水处理(40%土壤相对含水量持续14 d,然后恢复到70%土壤相对含水量维持7 d,RT)与对照(70%土壤相对含水量维持21 d,RCK)之间类黄酮合成途径中基因表达的差异。结果表明,干旱胁迫后,马铃薯中类黄酮合成途径变化显著(P0.01),该途径共有10个基因发生了差异表达,影响7个酶的合成,其中查尔酮合酶(EC 2.3.1.74)、柚皮素3-双加氧酶(EC 1.14.11.9)、类黄酮3′,5′-羟化酶(EC 1.14.13.88)的基因表达显著上调,从而可能促进了异黄酮、黄酮醇和黄酮以及高圣草素、二氢杨梅素的形成。PGSC0003DMG400019110和PGSC0003DMG400003563的上调表达导致查尔酮合酶和柚皮素3-双加氧酶的合成加快。复水对类黄酮合成途径的恢复效果较好,差异表达基因减少到2个,除莽草酸邻羟基肉桂酰转移酶(EC 2.3.1.133)与咖啡酰CoA-O-甲基转移酶(EC 2.1.1.104)的基因表达仍存在显著差异外,其余酶均已无差异。     

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     

中国水仙查尔酮合酶cDNA的克隆及序列分析（简报）

Chalcone synthase (CHS) is a key enzyme in the biosynthesis of all classes of flavonoids. The production of flower pigment is specifically regulated by the activity of CHS. We cloned the cDNA sequence of CHS-A gene from Narcissus by PCR and analyzed the coding sequence of gene. The result demonstrated that the sequence of the coding region was 1167bp, encoding a protein of 389 amino acid which was more than 80% homology with CHS of the other 8 plants, such as Nicotine abacus and Solana tuberosum.     

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。     

The chalcone synthase multigene family of Petunia hybrida (V30): sequence homology,chromosomal localization and evolutionary aspects

Chalcone synthase (CHS) genes in Petunia hybrida comprise a multigene family containing at least 7 complete members in the strain Violet 30 (V30). Based on a high sequence homology in both coding and non-coding sequence, a number of CHS genes can be placed into two subfamilies. By restriction fragment length polymorphism (RFLP) analysis it was shown that both chromosomes II and V carry one of these subfamilies, in addition to the other CHS genes identified so far. Members of a subfamily were found to be closely linked genetically. Analysis of the Petunia species that contributed to the hybrid nature of P. hybrida (P. axillaris, P. parodii, P. inflata and P. violacea) shows that none of the CHS gene clusters is specific for either one of the parents and therefore did not arise as a consequence of the hybridization. The number of CHS genes within a subfamily varies considerably among these Petunia species. From this we infer that the CHS subfamilies arose from very recent gene duplications.     

The chalcone synthase multigene family of Petunia hybrida (V30): sequence homology,chromosomal localization and evolutionary aspects

Chalcone synthase (CHS) genes in Petunia hybrida comprise a multigene family containing at least 7 complete members in the strain Violet 30 (V30). Based on a high sequence homology in both coding and non-coding sequence, a number of CHS genes can be placed into two subfamilies. By restriction fragment length polymorphism (RFLP) analysis it was shown that both chromosomes II and V carry one of these subfamilies, in addition to the other CHS genes identified so far. Members of a subfamily were found to be closely linked genetically. Analysis of the Petunia species that contributed to the hybrid nature of P. hybrida (P. axillaris, P. parodii, P. inflata and P. violacea) shows that none of the CHS gene clusters is specific for either one of the parents and therefore did not arise as a consequence of the hybridization. The number of CHS genes within a subfamily varies considerably among these Petunia species. From this we infer that the CHS subfamilies arose from very recent gene duplications.     

Characterization and structural features of a chalcone synthase mutation in a white-flowering line of Matthiola incanaR. Br. (Brassicaceae)

For Matthiola incana (Brassicaceae), used as a model system to study biochemical and genetical aspects of anthocyanin biosynthesis, several nearly isogenic colored wild type lines and white-flowering mutant lines are available, each with a specific defect in the genes responsible for anthocyanin production (genes e, f, and g). For gene f supposed to code for chalcone synthase (CHS; EC 2.3.1.74), the key enzyme of the flavonoid/anthocyanin biosynthesis pathway belonging to the group of type III polyketide synthases (PKS), the wild type genomic sequence of M. incana line 04 was determined in comparison to the white-flowering CHS mutant line 18. The type of mutation in the chs gene was characterized as a single nucleotide substitution in a triplet AGG coding for an evolutionary conserved arginine into AGT coding for serine (R72S). Northern blots and RT-PCR demonstrated that the mutated gene is expressed in flower petals. Heterologous expression of the wild type and mutated CHS cDNA in E. Scherichia coli, verified by Western blotting and enzyme assays with various starter molecules, revealed that the mutant protein had no detectable activity, indicating that the strictly conserved arginine residue is essential for the enzymatic reaction. This mutation, which previously was not detected by mutagenic screening, is discussed in the light of structural and functional information on alfalfa CHS and related type III PKS enzymes.