百合查尔酮合成酶基因的克隆与分析

以西伯利亚百合为试材,通过半巢式PCR和RT-PCR技术分别克隆了查尔酮合成酶基因(CHS)的DNA和cDNA.生物信息学分析显示,CHS的DNA序列全长1 397 bp(登录号HM622754),包含2个外显子和1个内含子;cDNA序列编码区全长1 182 bp(登录号HQ161731),编码393个氨基酸,具有3个典型的CHS蛋白结构域:N-末端结构域(Lys3-Pro229)、C-末端结构域(Gln239-Pro389)和聚合酶Ⅲ结构域(Met1-Thr391);不同百合品种的CHS基因编码的氨基酸序列相似性高达98%,表明百合CHS基因在进化上呈现出十分保守的趋势;不同植物CHS基因序列的系统进化邻接树结果表明:百合与单子叶植物鸢尾及禾本科的水稻、大麦、玉米等亲缘关系更为接近.     

苦荞查尔酮合成酶基因CHS的结构及花期不同组织表达量分析

采用同源克隆、染色体步移和RT-PCR技术,首次克隆到苦荞查尔酮合酶基因（CHS）的全长DNA序列和cDNA开放阅读框（ORF)序列. 序列分析表明,苦荞CHS DNA序列（GU172165）全长1 632 bp,含1个445 bp的内含子;cDNA编码区（HM852753）全长1 188 bp,编码395个氨基酸,命名为FtCHS. 生物信息学分析表明,FtCHS和推导的氨基酸序列与其它植物CHS基因同源率在95%以上,含有CHS多基因家族的标签序列（GFGPG）、活性位点、底物结合口袋位点和环化反应口袋位点. 半定量RT-PCR分析苦荞花期FtCHS空间表达模型表明,其表达量未成熟种子＞叶＞茎＞花＞根＞成熟种子,与苦荞芦丁含量的分布基本一致,具有组织特异性.     

金花茶查尔酮合成酶基因全长克隆与序列分析

利用RT-PCR和RACE方法,从我国珍稀植物金花茶(Camellia nitidissima)花瓣中获得了查尔酮合成酶(chalcone synthase,CHS)基因的cDNA全长,命名为Cn-CHS,GenBank登录号HQ269804.碱基序列分析表明,Cn-CHS全长1 454bp,包含77 bp的5'非翻译区、207 bp的3'非翻译区和一个长为1 170 bp编码389个氨基酸的开放阅读框.氨基酸序列分析显示该基因编码的蛋白具有CHS家族保守存在的所有功能活性位点和特征性多肽序列.氨基酸序列比对分析表明,CnCHS与蔷薇科、杜鹃花科、茄科等植物的CHS相似性都在92%以上;与山茶科山茶属物种山茶(C.japonica)CHS完全一致;与茶(C.sinensis)CHS相似性达99%,有5个氨基酸位点存在差异,其中包括一个功能性位点.     

化州柚查尔酮合成酶基因克隆与序列分析

利用CTAB-LiCl法提取高质量的化州柚总RNA,采用RT-PCR技术克隆查尔酮合成酶基因,获得广东道地药材化橘红资源化州柚的查尔酮合成酶基因。该基因编码区全长1176bp,编码391个氨基酸残基,与同样来源于柑橘属的查尔酮合成酶基因同源性高达98%。CTAB-LiCl法能提取高质量的化州柚总RNA,可以用于后续基因克隆和分析;克隆获得的查尔酮合成酶具有编码区,与同属植物相同基因具有高度序列同源性。     

苦荞和甜荞查尔酮合成酶基因的克隆及序列比较

以苦荞品种‘西农9920’和甜荞品种‘西农9976’为材料,根据其它植物查尔酮合成酶(chalcone synthase,CHS)基因DNA序列的保守区域设计的一对简并引物,进行PCR扩增,从2种荞麦基因组中克隆出了长度均为860 bp的CHS基因片段,对其进行回收、克隆,挑选阳性克隆测序;序列分析表明这2个片段含有CHS基因的N端和C端的结构域,分别为苦荞和甜荞的CHS基因片段,命名为FtCHS和FeCHS。对获得的2种荞麦CHS基因的DNA序列进行比较分析,发现两者间存在多达43处单碱基多态性,这些单碱基多态性可能是苦荞和甜荞种子中类黄酮含量差异的重要原因之一。苦荞和甜荞CHS与其它植物CHS的氨基酸序列的进化分析表明,其与同为蓼科的掌叶大黄和石竹科的满天星的同源性较近。     

大豆查尔酮合成酶(CHS)基因的克隆、表达及其在雪莲提取液中的代谢产物分析

以中国大豆为材料,利用PCR方法克隆查尔酮合成酶（Chalcone synthase,CHS）全基因,采用SOE法克隆得到去掉内含子的查尔酮合成酶基因,核酸序列分析表明,该基因编码区长1170bp,编码390个氨基酸,与已报道的CHS的cDNA序列同源率达到97%。构建pET-GMCHS工程表达质粒,通过大肠杆菌E.coli BL21（DE3）高效表达系统表达大豆CHS。 通过12% SDS-聚丙烯酰胺凝胶电泳分析表明,获得了分子量在42.9KD的一条蛋白质特异表达带。液相色谱分析大肠杆菌E.coli BL21（DE3）高效表达系统在雪莲提取液中的代谢产物,样品和空白对照样对比,样品在273nm,3.0min出现新的吸收峰,质谱分析结果表明CHS利用雪莲提取液中代谢中间产物合成了新的黄酮类物质。     

决明查尔酮合成酶基因的克隆及序列分析

以决明(Cassia tora)为实验材料,利用RT-PCR和RACE技术,从决明嫩叶中克隆出查尔酮合成酶(Chal-one synthase,CHS)基因,其cDNA全长为1 459 bp,编码一个由390个氨基酸残基组成的多肽.氨基酸序列分析表明,决明CHS基因的氨基酸序列中含有44.61%的中性疏水氨基酸,29.74%的中性亲水氨基酸,12.56%的酸性氨基酸和13.O8%的碱性氨基酸.决明CHS基因的氨基酸序列中具有CHS家族酶系的氨基酸保守残基,包括结合底物CoA的结合残基及催化聚酮合成的催化残基,表明其可能参与聚酮化合物的合成.决明与其它植物CHS的氨基酸序列的进化分析表明,其与同为豆科决明属的翼叶决明(Cassia alata)的同源性较近,并且CHS家族可以分为CHS亚家族与非CHS亚家族.将得到的序列提交GenBank,登录号为EU430077.     

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。     

苦荞中查尔酮合成酶全长基因的克隆及其序列分析

选用乌克兰伊琳娜苦荞为试验材料,以从叶片中提取的RNA为模板,应用RACE技术结合CODEHOP引物设计方法成功克隆出苦荞中查尔酮合成酶cDNA序列后,将其序列电子合并其全长后设计基因全长特异性引物.以DNA为模板进行PCR扩增出基因序列.通过生物信息学分析表明,该基因金长为1 906 bp,具有一个463 bp的内含子序列,编码区长度为1188 bp,编码395个氨基酸.Blastn序列比对发现本试验所获得的CHS基因序列与相近物种Rheum palmatum(登录号:DQ205352.1)的CHS基因同源性达86％.将得到的序列命名为RaCHS并提交GenBank,登录号为HQ434624.应用Clustalxl.81和MEGA4软件构建系统进化树,并进行了同源性分析.     

水蕨(Ceratopteris thalictroides)查尔酮合成酶基因的克隆和表达

查尔酮合酶(chalcone synthase, CHS)是植物类黄酮化合物合成的关键酶,有关蕨类植物CHS基因的序列及功能信息尚不完善。本研究采用快速扩增cDNA末端(RACE)技术克隆获得了模式蕨类植物——水蕨(Ceratopteris thalictroides)CtCHS基因(GenBank登录号:JX027616.1),其cDNA序列全长为1616 bp,具有3个外显子和2个内含子,开放阅读框(ORF)为1215 bp,编码404个氨基酸。进化树分析表明,CtCHS与问荆(Equisetum arvense)、松叶蕨(Psilotum nudum)和3种薄囊蕨的查尔酮合成酶基因聚为一枝,说明这些蕨类植物亲缘关系较近且为单系起源。通过构建原核表达体系成功获得CtCHS蛋白的多克隆抗体并用于免疫印迹分析,结果表明CtCHS基因的表达明显受紫外光(UV)诱导。CtCHS基因的克隆与表达分析为进一步研究水蕨类黄酮化合物的合成及其调控机制提供了依据。     

甜荞查尔酮合酶基因Chs的克隆及序列分析

利用RT-PCR技术从甜荞中克隆得到查耳酮合酶(CHS)的cDNA开放阅读框(ORF)序列,命名为FeChs,NCBI登录号为GU172166.1.该序列长1 179 bp,编码392个氨基酸,与其它植物CHS基因的同源性为78%～92%,其推导的氨基酸序列含有CHS高度保守的活性位点及CHS的标签序列GFGPG.     

Molecular cloning and expression profiling of a chalcone synthase gene from hairy root cultures of Scutellaria viscidula Bunge

A cDNA encoding chalcone synthase (CHS), the key enzyme in flavonoid biosynthesis, was isolated from hairy root cultures of Scutellaria viscidula Bunge by rapid amplification of cDNA ends (RACE). The full-length cDNA of S. viscidula CHS, designated as Svchs (GenBank accession no. EU386767), was 1649 bp with a 1170 bp open reading frame (ORF) that corresponded to a deduced protein of 390 amino acid residues, a calculated molecular mass of 42.56 kDa and a theoretical isoelectric point (pI) of 5.79. Multiple sequence alignments showed that SvCHS shared high homology with CHS from other plants. Functional analysis in silico indicated that SvCHS was a hydrophilic protein most likely associated with intermediate metabolism. The active sites of the malonyl-CoA binding motif, coumaroyl pocket and cyclization pocket in CHS of Medicago sativa were also found in SvCHS. Molecular modeling indicated that the secondary structure of SvCHS contained mainly α-helixes and random coils. Phylogenetic analysis showed that SvCHS was most closely related to CHS from Scutellaria baicalensis. In agreement with its function as an elicitor-responsive gene, the expression of Svchs was induced and coordinated by methyl jasmonate. To our knowledge, this is the first report to describe the isolation and expression of a gene from S. viscidula.     

Stress responses in alfalfa (Medicago sativa L.). 8. Cis-elements and trans-acting factors for the quantitative expression of a bean chalcone synthase gene promoter in electroporated alfalfa protoplasts

A chimeric gene consisting of a bean (Phaseolus vulgaris L.) chalcone synthase (CHS) promoter fused to a bacterial chloramphenicol acetyltransferase (CAT) reporter gene was strongly expressed, and further induced by fungal elicitor, when electroporated into alfalfa (Medicago sativa L.) suspension cell protoplasts. Functional analysis of 5 deletions of the CHS promoter-CAT construct in these protoplasts indicated that the region between –326 and –130 contained both activator and silencer elements. Co-electroporation experiments confirmed that these cis-acting elements were binding sites for functionally active trans factors. In vitro DNase I footprinting revealed four potential binding sites for alfalfa suspension cell nuclear proteins between positions –326 and –130 of the CHS promoter. These sites mapped to regions shown to contain functional cis-acting elements on the basis of the deletion analysis. Three of these sites mapped to previously identified binding sites for bean nuclear proteins. Competition gel retardation analysis using oligonucleotide probes containing binding site sequences revealed sequence-specific binding of alfalfa nuclear proteins to an AT-rich element and a putative GT-1 factor consensus binding sequence. Our results define cis elements and their cognate trans factors functionally active in determining the quantitative expression of a defense response gene in a heterologous transient expression system.Abbreviations CAT chloramphenicol acetyltransferase - CHS chalcone synthase (EC 2.3.1.74) - PAL L-phenylalanine ammonia-lyase (EC 4.3.1.5)     

Enhanced expression and differential inducibility of soybean chalcone synthase genes by supplemental UV-B in dark-grown seedlings

By developing gene-specific RT-PCR and using filters to allow transmission down to 290 nm (UV-B+) or blocking all radiation below 320 nm (UV-B–), the effect of UV-B+ and UV-B– light on expression of each of the presently known seven members of soybean chalcone synthase (CHS) gene family in dark-grown seedlings was analyzed. Dark expression was detectable already in 18 h dark-germinating embryos, with progressive increases on successive days, suggesting that chs belongs to a class of genes expressed very early during germination, and that the expression at this stage is either constitutive or induced by non-light-dependent factors present in the seed or made available following imbibition. Exposure of 18 h dark-germinating embryos to UV-B– or to UV-B+ light did not lead to an increase in chs signal. However, the 24 h dark-germinating embryos showed a distinct effect of UV-B+, interestingly coinciding with the stage when the head of seedlings was in the process of being pushed up above ground by stem elongation, suggesting the possibility of a developmental switch modulating the appearance of UV-B response. The response to UV-B– was most prominent in chs1 and almost silent in chs2, while the up-regulation by UV-B+ was most prominent in chs5 and chs6 and much less so in chs2. Interestingly, chs2 was noted to be the only member of the Gmchs gene family devoid of H-box, raising the possibility that the H-box may be a good indicator of the photo-inducibility of a chs gene.     

An unstable gene controlling developmental variegation in okra (Abelmoschus esculentus (L.) Moench)

Summary Genetic studies on radiation-induced chlorina and variegated mutants of okra (Abelmoschus esculentus (L.) Moench) revealed the existence of an unstable gene. The normal green color of the leaves is controlled by duplicate genes C₁ and C₂, either of which produces the green colour. The chlorina plants are C ₁ C ₁ C ₂ C ₂. The allele c ₁ ^v is dominant to both C ₁ and C ₂ but is unstable. The homozygote c ₁ ^v c ₁ ^v c ₂ c ₂ is a normal green while the heterozygote c _i ^v c ₁ c ₂ c ₂ has a variegated phenotype as a result of the mutation of c ₁ ^v to c ₁ during development. In green plants with a c ₁ ^v c{sh1/v}c ₂ c ₂ genotype, the autonomous mutation of one of the c ₁ ^v alleles to c ₁ may take place at the pre-meiotic stage. In the variegated genotype (c ₁ ^v c ₁ c ₂ c ₂), the mutation of c ₁ to c ₁ ^v may take place in early ontogeny, thus producing green plants. The allele C ₁, when associated with c ₁ ^v in a heterozygous condition, mutates to c ₁ at the pre-meiotic stage even in the presence of the allele C ₂.     

Low temperature enhances petunia flower pigmentation and induces chalcone synthase gene expression

Flower coloration is controlled by internal and external factors, including temperature. The aim of the present work was to examine the effect of temperature on anthocyanin synthesis and chalcone synthase gene ( chs ) expression in petunia flowers. A moderate-low temperature enhanced both anthocyanin accumulation and chs expression in the corollas. However, the effect on chs expression was not always correlated with that on anthocyanin content, suggesting a post-translational effect. The effect was local and required the exposure of corollas, but not the whole plant, to the ambient temperature. The response of chs to moderate-low temperatures did not coincide with its expression during flower development. Moderate-low temperatures only slightly affected gibberellic acid (GA₃)-induced chs expression in the light, but activated chs expression under non-inducing conditions, i.e. in the absence of GA₃ in the dark. The results of this study suggest that moderate-low temperatures do not simply enhance the developmental regulation of anthocyanin biosynthetic gene expression; they act as a specific and separate signal.     

Measurement and classification of genetic diversity in okra (Abelmoschus esculentus)

Eighteen accessions of okra of diverse ecological background were evaluated for genetic variability through the techniques of coefficient of racial likeness (CRL) and principal coordinate analysis (PCO). The variation patterns among the accessions were classified by using the techniques of metroglyph analysis and single-linkage cluster analysis. CRL and PCO produced similar results on the diversity of the accessions but the grouping of the accessions by metroglyph analysis and SLCA produced different results. The usefulness of metroglyph analysis depends on the amount of variation accounted for by the two principal characters on which the two co-ordinate axes are constructed. The techniques are compared.     

Signal transduction in the photocontrol of chalcone synthase gene expression*

The photocontrol of chalcone synthase gene expression was studied by means of promoter analyses, in vitro systems, photoreceptor mutants and microinjections, and pharmacological approaches. A 52 bp element of the promoter is necessary and sufficient to transfer light regulation. Chalcone synthase expression is primarily under the control of phyA and blue/UV photo receptors; the latter are functional even in the absence of phyA and phyB. Phytochromes seem to be soluble proteins and, within seconds of irradiation, light-dependent phosphorrylations were observed in membrane-depleted cytosol preparations, indicating very early processes of signal transduction. Microinjection and pharmacological experiments reveal that, in the phyA pathway, heterotrimeric G-proteins, cGMP and a genistein-sensitive protein kinase are involved, whereas the UV pathway includes several trimeric G-proteins and Ca/calmodulin-dependent steps.     

Expression of chalcone synthase and chalcone isomerase proteins in Arabidopsis seedlings

Antibodies have been developed against the first two enzymes of flavonoid biosynthesis in Arabidopsis thaliana. Chalcone synthase (CHS) and chalcone isomerase (CHI) were overexpressed and purified from Escherichia coli as fusion proteins with glutathione S-transferase from Schistosoma japonicum. The recombinant proteins were then used to immunize chickens and the resulting IgY fraction was purified from egg yolks. Immunoblots of crude protein extracts from Arabidopsis seedlings carrying wild-type and null alleles for CHS and CHI showed that the resulting antibody preparations provide useful tools for characterizing expression of the flavonoid pathway at the protein level. An initial analysis of expression patterns in seedlings shows that CHS and CHI proteins are present at high levels during a brief period of early seedling germination that just precedes the transient accumulation of flavonoid end-products.