CHS基因起源初探及其在被子植物中的进化分析

利用PCR与TAlL-PCR方法,从半月苔(Lunularia cructata(L.)Dum.ex Lindb)中获得了一段长约l 000 bp的基因片段,它与已知的CHS基因在核苷酸水平上的相似性大于56%,在氨基酸水平上的相似性大于60%,所推断的氨基酸序列中酶反应的4个催化位点与已知晶体结构的紫花苜蓿MCHS2A上的催化位点相同,首次证明了苔类植物中可能存在类CHS基因,将CHS基因的起源时间推到苔藓类植物出现之前.以该序列和两种蕨类植物(Psilotumnudum(L.)Griseb.和Equisetum arvense L.)的CHS序列作为外类群,应用邻接法、最大简约法和最大似然法分别构建了被子植物的CHS的分子系统树.结果表明,大部分科中的CHS分布在不同的分支上,而十字花科、可科和禾本科各自聚成一个单系类群.以邻接树为依据,对茄科、旋花科和菊科的CHS基因进行了相对碱基替换速率的检测,发现这三个科内或科间序列的替换速率不一致.被子植物的CHS基因在基因拷贝数目、碱基替换速率以及重复/丢失事件的发生上都存在较大的差异,这种差异可能与被子植物的生活史、生活环境、花的特性以及对外界的防御系统等的多样性相关.     

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.     

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.     

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。     

真双子叶基部类群十种植物的CHS基因家族成员分析

用PCR方法从目前极少被报道的真双子叶基部类群10种植物的总DNA中扩增出CHS基因外显子2的部分序列进行分析,经克隆后测序,共得到26个不同的片段。分析表明,所有序列具有较高的同源性（〉70％）,每种植物有2—4个不同的拷贝,在金鱼藻中发现有一个拷贝（CedeCHS3）含有一段长29个碱基的缺失,可能已失去了该基因的功能,而来自银桦的GrroCHS8拷贝具有较多活性位点的变异,可能具有了新的功能。在对碱基含量的分析中表明,只有来自雀舌黄杨的序列有一定的GC偏好,特别是第三位的GC含量达70％以上。用贝叶斯法、最大简约法和邻接法构建的CHS基因的分子系统树均由3个主要分支构成,其中一个分支由来自金鱼藻的4个序列聚在一起构成了subfamilyⅡ,来自昆栏树的2个序列聚在一起网接于subfamilyⅢ中;来自独叶草的3个序列分散于同一亚家族（subfamilyⅠ）的不同分支中;而多数种的序列分散在相距较远的两个分支中,构成了两个主要的亚家族subfamilyⅠ、subfamilyⅢ。从所建分子系统树看真双子叶基部类群植物的CHS基因家族成员来源于该类群形成前的两个祖先拷贝,在不同植物这两个祖先拷贝经历了不同的进化过程,这些进化上的差异可能与不同植物的生活习性及生存环境的多样性相关。     

基于psaA和psbA基因的红索藻目系统发育研究

对红索藻目植物棘刺红索藻(Thorea hispida)的psaA和psbA基因进行扩增和测序,并与其它类群比对分析,分别用最大简约法、邻接法和贝叶斯法构建系统发育树。结果显示,psaA和psbA基因测得的序列片段分别为825和920 bp,psaA基因A、T、C、G碱基含量分别为29.9%、35.8%、16.5%和17.8%,psbA基因A、T、C、G碱基含量分别为27.5%、35.3%、16.8%和20.4%,两个基因A+T含量均高于C+G含量,说明两个基因在进化上均有碱基的偏好性。用3种方法所构建的系统树拓扑结构基本一致,红索藻目植物均聚合于一个分支,独立于其它类群,支持红索藻目为一独立的目。     

苦荞查尔酮合酶基因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。     

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.     

二倍体蔷薇FT基因序列多样性研究

以3个类群73个二倍体蔷薇属(Rosa)植物为材料,克隆获得其FLOWERING LOCUS T(FT)同源基因,并对该基因的编码区序列进行多态性分析以及多维尺度(MDS)聚类分析。结果显示,73个二倍体蔷薇植物的FT基因共检测到215个核苷酸多态性位点,其中包括214个SNP和1个缺失突变,平均185个碱基发生1次突变;氨基酸多态性分析结果显示共有35个氨基酸发生变异,平均379.6个氨基酸残基发生1次突变;突变位点统计分析结果发现39、258、426 bp位点是高频突变位点,其碱基由A或C突变为T。MDS聚类分析结果表明,3个类群FT基因编码区序列的碱基组内差异依次排序为:野生种月季组中国古老月季,氨基酸组内差异依次排序为:中国古老月季月季组野生种,推测中国古老月季在长期栽培驯化过程中,其FT基因可能经历了较强的人工选择压力,月季组的种和变种可能是古老月季的重要亲本来源。     

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.     

Cloning and characterization of chalcone synthase from the moss, Physcomitrella patens

Since the early evolution of land plants from primitive green algae, flavonoids have played an important role as UV protective pigments in plants. Flavonoids occur in liverworts and mosses, and the first committed step in the flavonoid biosynthesis is catalyzed by chalcone synthase (CHS). Although higher plant CHSs have been extensively studied, little information is available on the enzymes from bryophytes. Here we report the cloning and characterization of CHS from the moss, Physcomitrella patens. Taking advantage of the available P. patens EST sequences, a CHS (PpCHS) was cloned from the gametophores of P. patens, and heterologously expressed in Escherichia coli. PpCHS exhibited similar kinetic properties and substrate preference profile to those of higher plant CHS. p-Coumaroyl-CoA was the most preferred substrate, suggesting that PpCHS is a naringenin chalcone producing CHS. Consistent with the evolutionary position of the moss, phylogenetic analysis placed PpCHS at the base of the plant CHS clade, next to the microorganism CHS-like gene products. Therefore, PpCHS likely represents a modern day version of one of the oldest CHSs that appeared on earth. Further, sequence analysis of the P. patens EST and genome databases revealed the presence of a CHS multigene family in the moss as well as the 3'-end heterogeneity of a CHS gene. Of the 19 putative CHS genes, 10 genes are expressed and have corresponding ESTs in the databases. A possibility of the functional divergence of the multiple CHS genes in the moss is discussed.     

Starter substrate specificities of wild-type and mutant polyketide synthases from Rutaceae

Chalcone synthases (CHSs) and acridone synthases (ACSs) belong to the superfamily of type III polyketide synthases (PKSs) and condense the starter substrate 4-coumaroyl-CoA or N-methylanthraniloyl-CoA with three malonyl-CoAs to produce flavonoids and acridone alkaloids, respectively. ACSs which have been cloned exclusively from Ruta graveolens share about 75-85% polypeptide sequence homology with CHSs from other plant families, while 90% similarity was observed with CHSs from Rutaceae, i.e., R. graveolens, Citrus sinensis and Dictamnus albus. CHSs cloned from many plants do not accept N-methylanthraniloyl-CoA as a starter substrate, whereas ACSs were shown to possess some side activity with 4-coumaroyl-CoA. The transformation of an ACS to a functional CHS with 10% residual ACS activity was accomplished previously by substitution of three amino acids through the corresponding residues from Ruta-CHS1 (Ser132Thr, Ala133Ser and Val265Phe). Therefore, the reverse triple mutation of Ruta-CHS1 (mutant R2) was generated, which affected only insignificantly the CHS activity and did not confer ACS activity. However, competitive inhibition of CHS activity by N-methylanthraniloyl-CoA was observed for the mutant in contrast to wild-type CHSs. Homology modeling of ACS2 with docking of 1,3-dihydroxy-N-methylacridone suggested that the starter substrates for CHS or ACS reaction are placed in different topographies in the active site pocket. Additional site specific substitutions (Asp205Pro/Thr206Asp/His207Ala or Arg60Thr and Val100Ala/Gly218Ala, respectively) diminished the CHS activity to 75-50% of the wild-type CHS1 without promoting ACS activity. The results suggest that conformational changes in the periphery beyond the active site cavity volumes determine the product formation by ACSs vs. CHSs in R. graveolens. It is likely that ACS has evolved from CHS, but the sole enlargement of the active site pocket as in CHS1 mutant R2 is insufficient to explain this process.     

Structure of chalcone synthase and the molecular basis of plant polyketide biosynthesis.

Chalcone synthase (CHS) is pivotal for the biosynthesis of flavonoid antimicrobial phytoalexins and anthocyanin pigments in plants. It produces chalcone by condensing one p-coumaroyl- and three malonyl-coenzyme A thioesters into a polyketide reaction intermediate that cyclizes. The crystal structures of CHS alone and complexed with substrate and product analogs reveal the active site architecture that defines the sequence and chemistry of multiple decarboxylation and condensation reactions and provides a molecular understanding of the cyclization reaction leading to chalcone synthesis. The structure of CHS complexed with resveratrol also suggests how stilbene synthase, a related enzyme, uses the same substrates and an alternate cyclization pathway to form resveratrol. By using the three-dimensional structure and the large database of CHS-like sequences, we can identify proteins likely to possess novel substrate and product specificity. The structure elucidates the chemical basis of plant polyketide biosynthesis and provides a framework for engineering CHS-like enzymes to produce new products.     

山茶属CHS基因家族的组成和分子进化初探

用PCR方法从4种山茶属(Camellia)(山茶科)(Tlaeaceae)植物的总DNA中分别扩增到CHS基因外显子2的部分序列,经克隆、测序得到16个该基因的序列,这些序列与来自GenBank的该属另一种植物的3个序列及作为外类群的大豆(Glycine max (L)Merr)的2个序列一起进行分析。研究表明,山茶属CHS基因家族在进化过程中已分化为A、B、c三个亚家族,包括A1、A2、A3、B1、B2、C等6类不同的基因成员;其中只有A2类成员为全部被研究的5种植物所共有,而其他5类成员只在部分被研究的植物中发现;所有这些CHS成员具有很高的同源性：在核苷酸水平上同一亚家族内基本上高于90％,不同亚家族间也在78％以上。从推测的氨基酸组成看,山茶属内CHS基因的功能已发生了分化,各类成员的碱基替代率有较大差异;从分子系统发育树和可能的氨基酸组成分析,山茶属具有新功能的基因成员是在经过基因重复后,或是由少数几个位点的突变而成,或是由逐渐积累的突变而形成的。进一步分析认为,该属CHS基因的分化直到近期还在活跃地进行,并且不同种的进化式样有一定的差别,这种不同的进化式样可能是物种形成后受不同环境因素影响而形成的。     

Isolation and sequence analysis of a chalcone synthase cDNA ofMatthiola incana R. Br. (Brassicaceae)

A cDNA clone (pcM12) of the chalcone synthase (CHS) ofMatthiola incana R. Br. (Brassicacease) was isolated from a cDNA library, sequenced and analysed. It comprises the complete coding sequence for the CHS and 5 and 3 untranslated regions. The deduced amino acid sequence shows that theMatthiola incana CHS consists of 394 amino acid residues. Comparison with CHS amino acid sequences of other plants indicates more than 82% homology.     

甘蔗ACC氧化酶基因片段的克隆与序列分析

1- 氨基环丙烷-1-羧酸（ACC）氧化酶是植物乙烯合成的一个关键酶,乙烯作为一种内源激素,对植物生长、老熟过程有多方面的调节作用。根据报道的各种植物ACC氧化酶氨基酸序列上前后两个保守区设计两个简并引物,以甘蔗总DNA为模板,通过PCR扩增到一个940bp的基因片段。将片段序列在MCBI的BLAST软件上进行同源性搜寻,显示的63个序列全部是ACC氧化酶基因,因而认为克隆到的片段就是甘蔗ACC氧化酶基因的一个成员。经对不同植物来源的ACC氧化酶基因家族进行比较分析,去除一个103bp的“内含子“后,推导的氨基酸序列为279个残基,占推测全长氨基酸残基总数的86％左右。经同源性分析,序列与毛竹和水稻ACC氧化酶的同源率达到86％。系统进化分析表明,该序列最先与水稻、其次和香蕉的ACC氧化酶聚类,然后再与双子叶植物的ACC氧化酶聚类,符合按形态特征分类的血缘关系。基因的获得对下一步了解乙烯的合成表达与甘蔗生长、成熟过程之间的关系奠定了基础。     

Type I and Type II genes for the chlorophyll a/b-binding protein in the gymnosperm Pinus sylvestris (Scots pine): cDNA cloning and sequence analysis

A cDNA library was constructed from mRNA prepared from light-treated seedlings of Scots pine (Pinus sylvestris L.) and cDNAs for the chlorophyll a/b-binding protein LHC-II were identified using a pea gene as the heterologous probe. Three cDNA clones were sequenced. The deduced amino acid sequences of two of the genes corresponded to Type I and one to Type II LHC-II proteins which were ca. 90% homologous to their angiosperm counterparts. The transit peptides of the Scots pine preLHC-II showed features common to angiosperm transit peptides. The three cDNAs had a 70 to 75% preference for G+C in the third base position. CpG and GpC profiles and degenerate codon position bias suggested that two of the corresponding genes lie within CpG islands.     

Structure of the <Emphasis Type="Italic">TADHN</Emphasis> gene for dehydrin-like protein of soft wheat and activation of its expression by ABA and 24-epibrassinolide

The structure of the cloned fragment of wheat (Triticum aestivum L.) TADHN gene encoding dehydrin-like protein was examined. A comparative analysis of nucleotide and deduced amino acid sequences revealed a high homology of this fragment with sequences of the barley dhn8 gene and wheat wcor gene family. In deduced amino acid sequence of the TADHN fragment, a 15-residue region EKKGFLEKIKEKLPG was found, which corresponded to a highly conserved K-segment of dehydrins. Wheat seedling treatment with 3.7 μM ABA and 0.4 μM 24-epibrassinolide exerted similar stimulatory effects on expression of the TADHN gene, which indicates the involvement of dehydrins in the protective action of these phytohormones in wheat plants.     

Transformation of acridone synthase to chalcone synthase.

Acridone synthase (ACS) and chalcone synthase (CHS) catalyse the pivotal reactions in the formation of acridone alkaloids or flavonoids. While acridone alkaloids are confined almost exclusively to the Rutaceae, flavonoids occur abundantly in all seed-bearing plants. ACSs and CHSs had been cloned from Ruta graveolens and shown to be closely related polyketide synthases which use N-methylanthraniloyl-CoA and 4-coumaroyl-CoA, respectively, as the starter substrate to produce the acridone or naringenin chalcone. As proposed for the related 2-pyrone synthase from Gerbera, the differential substrate specificities of ACS and CHS might be attributed to the relative volume of the active site cavities. The primary sequences as well as the immunological cross reactivities and molecular modeling studies suggested an almost identical spatial structure for ACS and CHS. Based on the Ruta ACS2 model the residues Ser132, Ala133 and Val265 were assumed to play a critical role in substrate specificity. Exchange of a single amino acid (Val265Phe) reduced the catalytic activity by about 75% but grossly shifted the specificity towards CHS activity, and site-directed mutagenesis replacing all three residues by the corresponding amino acids present in CHS (Ser132Thr, Ala133Ser and Val265Phe) fully transformed the enzyme to a functional CHS with comparatively marginal ACS activity. The results suggested that ACS divergently has evolved from CHS by very few amino acid exchanges, and it remains to be established why this route of functional diversity has developed in the Rutaceae only.     

甘薯NBS类抗病基因类似物的分离与序列分析

利用已克隆植物抗病基因NBS（Nucleotide binding site）序列中的保守模体（motif）“P-loop”和“GLPL”合成简并引物,以甘薯（Ipomoea batatas）栽培品种青农2号基因组DNA为模板进行PCR扩增,通过T/A克隆、测序和序列分析,共得到15条具有连续ORF的抗病基因类似物（Resistance gene analogues,RGAs）序列,它们之间核苷酸序列间的相似性系数在41.2%-99.4%之间,而相应推测的氨基酸序列间的相似性系数在20.6%-100%之间,同时对分离的RGAs的核苷酸和氨基酸序列进行系统发育树分析,表明甘薯RGAs可分为TIR（Drosophila Toll or human interleukin receptor-like）和nonTIR两类.对甘薯RGAs和5个已克隆植物NBS的氨基酸序列进行结构分析表明,它们包括“P-loop”、“Kinase-2”、“Kinase-3a”、“GLPL”4个抗病基因所共有的保守模体.这些表明甘薯与其它物种的NBS类RGAs可能具有同样的起源和进化机制.