大豆KNOX基因家族的结构和表达分析

KNOX基因家族编码同源异型盒蛋白, 在植物生长发育过程中起重要调控作用。利用生物信息学手段在全基因组水平上对大豆(Glycine max)KNOX家族基因进行鉴定和分类, 并分析其基因结构、蛋白同源结构域特征以及基因表达方式。研究结果表明: 大豆中的27个GmKNOX基因可以分为GmKNOX I和GmKNOX II两个亚类, 其中GmKNOX I类可分为3个主要的进化支, GmKNOX II类分为2个主要的进化支; 26个GmKNOX基因不均匀地分布在16条染色体上, GmKNOX27尚无法定位。不同组织表达谱的分析表明: GmKNOX I类基因表达部位比较集中, 以茎顶端分生组织中表达量最高; 而GmKNOX II类基因的表达特异性较GmKNOX I类低, 表达部位更广泛。     

百脉根TALE转录因子家族的鉴定与生物信息学分析

三胺酸环延伸(TALE)蛋白是一类在植物生长发育过程中调控分生组织分化的转录因子。本研究通过生物信息学手段从豆科模式植物百脉根(Lotus japonicus(Regel)K.Larsen)全基因组中筛选出分布于6条染色体上的40条TALE家族基因,并对其保守结构域、基因结构、系统进化、在染色体上的分布、理化性质以及部分典型基因的组织表达差异等进行分析。根据结构域不同可将百脉根TALE家族分为BELL和KNOX两个亚族;百脉根TALE家族在进化上较为保守,分化上与大豆存在较大差异;该家族基因有外显子4～6个,氨基酸序列长度在271～792之间,家族成员蛋白均为弱酸性蛋白。Realtime PCR分析表明该家族基因表达与motif元件数之间存在相关性;BELL亚族主要在顶芽表达,KNOX亚族则主要在根组织中表达。研究结果为进一步克隆百脉根TALE基因和分析其功能奠定基础。     

百脉根TALE转录因子家族的鉴定与生物信息学分析北大核心CSCD

三胺酸环延伸(TALE)蛋白是一类在植物生长发育过程中调控分生组织分化的转录因子。本研究通过生物信息学手段从豆科模式植物百脉根(Lotus japonicus(Regel)K.Larsen)全基因组中筛选出分布于6条染色体上的40条TALE家族基因,并对其保守结构域、基因结构、系统进化、在染色体上的分布、理化性质以及部分典型基因的组织表达差异等进行分析。根据结构域不同可将百脉根TALE家族分为BELL和KNOX两个亚族;百脉根TALE家族在进化上较为保守,分化上与大豆存在较大差异;该家族基因有外显子4～6个,氨基酸序列长度在271～792之间,家族成员蛋白均为弱酸性蛋白。Realtime PCR分析表明该家族基因表达与motif元件数之间存在相关性;BELL亚族主要在顶芽表达,KNOX亚族则主要在根组织中表达。研究结果为进一步克隆百脉根TALE基因和分析其功能奠定基础。     

关于青岛文昌鱼SoxB2和SoxC基因的研究：进化保守性分析

大多数Sox基因在细胞命运决定和分化过程中起着重要的作用.本研究分离了青岛文昌鱼（Branchiostoma belcheri）的SoxB2和SoxC基因,对其预测的蛋白序列进行了序列比对、进化树分析以及基因时空表达分析.结果显示,文昌鱼SoxB2和SoxC虽然在进化树中不属于脊椎动物SoxB2和SoxC进化支,但它们在进化过程中基因的结构和表达部位保守性很高,都具有保守的HMG结构域;这两个基因在胚胎发育早期都在神经外胚层和原肠腔壁表达,而在成体中不仅存在于神经索中,还存在于肠、肝盲囊、鳃和卵母细胞中.SoxB2和SoxC基因表达部位的相似说明两者可能在中枢神经系统、性腺及免疫系统的胚胎发育和成体的免疫应答过程中共同起作用;两者与脊椎动物同源基因的表达部位相同说明这两个基因在进化过程中可能功能保守.     

麻竹同源异型盒基因DlKNOX的克隆及表达分析

为探讨同源异型盒(KNOX)基因在麻竹(Dendrocalamus latiflorus)茎秆发育中的作用,采用RT-PCR和RACE技术,从其幼茎中克隆了1个KNOX同源基因,命名为Dl KNOX,其c DNA序列全长为1511 bp,包含5′UTR 196 bp、3′UTR 238 bp和编码区1077 bp。该基因编码含358氨基酸的蛋白,具有KNOX1、KNOX2、ELK和Homeobox KN等4个保守结构域,符合KNOX家族的特征,属于I类蛋白。生物信息学分析表明,该基因编码的蛋白与水稻OSH1的一致性最高(86%)。组织表达特异性分析表明,Dl KNOX在节部的表达丰度最高,其次为幼茎,根中最低。Dl KNOX基因在大肠杆菌(Escherichia coli)中经诱导表达,获得1条分子量约为82 k Da的重组蛋白,与预期的重组蛋白分子量一致(包含了MBP标签蛋白42.5 k Da和Dl KNOX蛋白39.5 k Da)。该基因在大肠杆菌中的最适表达条件为28℃,0.3 mmol L–1 IPTG诱导2 h。这为进一步研究Dl KNOX在麻竹茎秆发育中的功能奠定了基础。     

绿豆VrWOX基因家族鉴定及表达分析北大核心CSCD

WOX(WUSCHEL-related homebox)基因家族是植物特有的一类转录因子,是同源盒(homeobox,HB)转录因子超家族中的重要成员。WOX基因在植物干细胞调节及生殖发育过程中具有重要作用,其功能已在多个植物物种中鉴定。然而绿豆(Vigna radiate)VrWOX基因家族信息尚不清楚。本研究通过同源比对和聚类分析,在绿豆基因组中鉴定了42个VrWOX基因。VrWOX基因在绿豆染色体中分布不均,其中7号染色体含有的VrWOX数量最多。VrWOX基因分为古老进化支(19个VrWOX)、中等进化支(12个VrWOX)和年轻进化支(WUSCHEL进化支,11个VrWOX)3个亚类。种内和种间共线性分析发现,VrWOX基因共有12个重复事件,与拟南芥(Arabidopsis thaliana)AtWOX有15个同源基因对,与菜豆(Phaseolus vulgaris)PvWOX有22个同源基因对。VrWOX基因在基因结构、保守基序等方面存在很大差异,因而可能存在功能差异。VrWOX基因启动子区域含有不同种类和不同数量的顺式作用元件,导致VrWOX基因在不同组织中表现出不同的基因表达模式。本研究对VrWOX基因家族信息和表达模式进行了分析,为绿豆VrWOX基因功能和调控网络的解析奠定了一定的理论依据。     

蕙兰CyfaSTK基因的克隆与表达分析

采用同源克隆的方法,从蕙兰（Cymbidium faberi Rolfe）花芽中克隆获得CyfaSTK基因的cDNA序列,并对其进行生物信息学分析及基因表达分析。结果显示,该基因全长843 bp,其中开放阅读框（ORF）长705 bp,共编码234个氨基酸和1个终止密码子。同源蛋白序列比对及分子系统发育分析结果表明,CyfaSTK蛋白属于D类MADS-box转录因子STK-like进化系,含有MADS、I、K和C等4个结构域,其C末端转录激活区含有2个保守的基元：AG motifⅠ和AG motifⅡ,此外,还具有一个在天门冬目植物中相对保守的基元MD motif。基因表达的组织特异性分析结果显示：蕙兰CyfaSTK基因在花萼、花瓣、唇瓣、药帽、子房中均有表达,但在叶片中不表达,其中在子房中的表达量与其他组织相比,差异达到极显著水平;CyfaSTK在花芽经过休眠后的萌动期表达量最高,且在开花当天该基因表达量有上升趋势。研究结果表明CyfaSTK基因不仅参与调控蕙兰花器官的发育过程,且对子房及合蕊柱的正常发育具有重要作用。     

樱胚珠发育调控基因PrseSTK在单瓣与重瓣花中的表达比较

用同源克隆方法,从日本晚樱（Prunus lannesiana）花芽中克隆出了PrseSTK基因的cDNA全长,GenBank登录号为GU332504。其包括1个共669 bp的完整开放阅读框,编码222个氨基酸和1个终止密码子。同源序列比对和分子系统进化分析表明,PrseSTK是拟南芥的STK同源基因,其编码蛋白的C末端拥有2个高度保守模体：AG motif Ⅰ和Ⅱ,属D类MADS-box转录因子。其在花器官中表达的组织特异性分析表明,在单瓣‘大岛樱’中,PrseSTK主要在雄蕊和雌蕊中表达;但在重瓣品种‘普贤像’中,其在萼片、雄蕊和叶化雌蕊中均有表达。其在2个品种4轮花器官中的表达呈现明显的差异,并与拟南芥STK基因表达的组织特异性也有一定的差别;其在花萼中的异位表达可能与重瓣品种萼筒异位子房的发育调控相关。     

蒺藜苜蓿花器官特异基因的表达分析

蒺藜苜蓿(Medicago truncatula G.)花器官特异表达基因是参与其花器官形成与发育的重要基因。筛选蒺藜苜蓿的花器官特异表达基因, 寻找这类基因在其他模式植物中的直系同源基因, 并将其表达模式在不同植物间进行比较, 有利于深入的理解这类基因在蒺藜苜蓿花器官发育中的功能。根据蒺藜苜蓿表达谱, 并以其PISTILLATA(PI)基因为模板, 文章筛选了97个蒺藜苜蓿花器官特异表达基因(Ratio≥10, 且Z≥7.9)。通过同源比对, 确定了这类基因在拟南芥(Arabidopsis thaliana L.)、大豆(Glycine max L.)、百脉根(Lotus japonicus L.)和水稻(Oryza sativa L.)中的直系同源基因。对这类基因在5种植物中的表达量、表达部位和功能进行比较, 发现进化关系较近的植物, 直系同源基因的表达变异较小, 而进化关系较远的植物, 直系同源基因的表达变异较大。进一步对表达分化较大的直系同源基因进行启动子分析, 发现不同植物中直系同源基因表达模式的变化与启动子中调控元件的特性有关。     

蒺藜苜蓿花器官特异基因的表达分析

蒺藜苜蓿(Medicago truncatula G)花器官特异表达基因是参与其花器官形成与发育的重要基因。筛选蒺藜苜蓿的花器官特异表达基因,寻找这类基因在其他模式植物中的直系同源基因,并将其表达模式在不同植物间进行比较,有利于深入的理解这类基因在蒺藜苜蓿花器官发育中的功能。根据蒺藜苜蓿表达谱,并以其PISTILLAZA(PI)基因为模板,文章筛选了97个蒺藜苜蓿花器官特异表达基因(Ratio≥10,且Z≥7.9).通过同源比对,确定了这类基因在拟南芥(Arabidopsis thaliana L.)、大豆(Glycinemax L.)、百脉根(Lotusjaponicus L.)和水稻(Oryzasativa L.)中的直系同源基因。对这类基因在5种植物中的表达量、表达部位和功能进行比较,发现进化关系较近的植物,直系同源基因的表达变异较小,而进化关系较远的植物,直系同源基因的表达变异较大。进一步对表达分化较大的直系同源基因进行启动子分析,发现不同植物中直系同源基因表达模式的变化与启动子中调控元件的特性有关。     

KNOX homeobox genes potentially have similar function in both diploid unicellular and multicellular meristems, but not in haploid meristems

Members of the class 1 knotted-like homeobox (KNOX) gene family are important regulators of shoot apical meristem development in angiosperms. To determine whether they function similarly in seedless plants, three KNOX genes (two class 1 genes and one class 2 gene) from the fern Ceratopteris richardii were characterized. Expression of both class 1 genes was detected in the shoot apical cell, leaf primordia, marginal part of the leaves, and vascular bundles by in situ hybridization, a pattern that closely resembles that of class 1 KNOX genes in angiosperms with compound leaves. The fern class 2 gene was expressed in all sporophyte tissues examined, which is characteristic of class 2 gene expression in angiosperms. All three CRKNOX genes were not detected in gametophyte tissues by RNA gel blot analysis. Arabidopsis plants overexpressing the fern class 1 genes resembled plants that overexpress seed plant class 1 KNOX genes in leaf morphology. Ectopic expression of the class 2 gene in Arabidopsis did not result in any unusual phenotypes. Taken together with phylogenetic analysis, our results suggest that (a) the class 1 and 2 KNOX genes diverged prior to the divergence of fern and seed plant lineages, (b) the class 1 KNOX genes function similarly in seed plant and fern sporophyte meristem development despite their differences in structure, (c) KNOX gene expression is not required for the development of the fern gametophyte, and (d) the sporophyte and gametophyte meristems of ferns are not regulated by the same developmental mechanisms at the molecular level.     

Revelation of ancestral roles of KNOX genes by a functional analysis of <Emphasis Type="Italic">Physcomitrella</Emphasis> homologues

KNOX genes are indispensable elements of indeterminate apical growth programmes of vascular plant sporophytes. Since little is known about the roles of such genes in non-vascular plants, functional analysis of moss KNOX homologues (MKN genes) was undertaken using the genetically amenable model plant, Physcomitrella patens. Three MKN genes were inactivated by targeted gene knockout to produce single, double and triple mutants. MKN2 (a class 1 KNOX gene) mutants were characterised by premature sporogenesis, abnormal sporophyte ontogeny and irregular spore development. MKN4 (a second class 1 gene) mutants were phenotypically normal. MKN1-3 (a class 2 KNOX gene) mutants exhibited defects in spore coat morphology. Analysis of double and triple mutants revealed that the abnormal sporophytic phenotype of MKN2 mutants was accentuated by mutating MKN4 and to a lesser degree by mutating MKN1-3. The aberrant spore phenotype of MKN1-3 and MKN2 mutants was exacerbated by mutating MKN4. This study provides the first instance in which an abnormal phenotype has been associated with the disruption of a class 2 KNOX gene as well as the first demonstrated case of functional redundancy between a class 1 and a class 2 KNOX gene. We conclude that KNOX genes play significant roles in programming sporophytic development in moss and we provide evidence that ancestral function(s) of this gene family were instrumental in the successful transition of plants to a terrestrial environment.     

Phylogenetic relationships and evolution of the KNOTTED class of plant homeodomain proteins.

Knotted-like (KNOX) proteins constitute a group of homeodomain proteins involved in pattern formation in developing tissues of angiosperms and other green plants. We conducted phylogenetic analyses of nucleotide and amino acid sequences of all known KNOX proteins in order to examine their evolution. Our analyses reveal two groups of KNOX proteins, classes I and II. Dicot and monocot sequences occur in both classes, indicating that the protein classes arose prior to the origin of the monocots. A conifer (Picea) sequence is nested within class I, suggesting that there are likely to be other copies of KNOX genes in this and other conifers. The orthology of several grass genes (including Zea Kn1, ZMKN1) is strongly supported by phylogenetic and synteny analyses. However, no compelling evidence supports the hypothesis of orthology previously proposed for several dicot genes and ZMKN1. Analysis of expression patterns suggests that the ancestral KNOX gene was expressed in all plant parts and that the propensity to be downregulated in roots and leaves evolved in the class I genes.     

Divergence and differential expression of soybean actin genes

DNA sequence analysis as well as genomic blotting experiments using cloned soybean actin DNA sequences as probes show that large sequence heterogeneity exists among members of the soybean actin multigene family. This heterogeneity suggested that the members of this family might be diverged in function and/or regulation. Five of the six soybean actin gene family members examined are shown to be significantly more diverged from one another than members of other known actin gene families. This high level of divergence was utilized in the preparation of actin gene-specific probes in the analysis of the complexity and expression of these members of the soybean actin gene family. Hybridization studies indicate that the six soybean actin genes fall into three classes with a pair of genes in each class. These six genes account for all but two actin gene fragments detected in the soybean genome. We have compared the relative steady state mRNA levels of these classes of soybean actin genes in three organs of soybean. We find that actin genes SAc6 and SAc7 are most highly expressed accounting for 80% of all actin mRNA with respect to the six soybean actin genes examined. Actin genes SAc3 and SAc1 are expressed at intermediate and low levels respectively; and SAc2 and SAc4 are expressed at barely detectable levels. Four of the six soybean actin genes appear to be expressed at the same level in root, shoot and hypocotyl. SAc3 and SAc7 genes appear to be more highly expressed in shoot and 2,4-dichlorophenoxyacetic acid-induced hypocotyl than in root and hypocotyl.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of the alternative oxidase promoters from soybean

Alternative oxidase (Aox) is a nuclear-encoded mitochondrial protein. In soybean (Glycine max), the three members of the gene family have been shown to be differentially expressed during normal plant development and in response to stresses. To examine the function of the Aox promoters, genomic fragments were obtained for all three soybean genes: Aox1, Aox2a, and Aox2b. The regions of these fragments immediately upstream of the coding regions were used to drive beta-glucuronidase (GUS) expression during transient transformation of soybean suspension culture cells and stable transformation of Arabidopsis. The expression patterns of the GUS reporter genes in soybean cells were in agreement with the presence or absence of the various endogenous Aox proteins, determined by immunoblotting. Deletion of different portions of the upstream regions identified sequences responsible for both positive and negative regulation of Aox gene expression in soybean cells. Reporter gene analysis in Arabidopsis plants showed differential tissue expression patterns driven by the three upstream regions, similar to those reported for the endogenous proteins in soybean. The expression profiles of all five members of the Arabidopsis Aox gene family were examined also, to compare with GUS expression driven by the soybean upstream fragments. Even though the promoter activity of the upstream fragments from soybean Aox2a and Aox2b displayed the same tissue specificity in Arabidopsis as they do in soybean, the most prominently expressed endogenous genes in all tissues of Arabidopsis were of the Aox1 type. Thus although regulation of Aox expression generally appears to involve the same signals in different species, different orthologs of Aox may respond variously to these signals. A comparison of upstream sequences between soybean Aox genes and similarly expressed Arabidopsis Aox genes identified common motifs.