白桦BpSPL2基因启动子的克隆及表达分析

SPL(SQUAMOSA promoter-binding protein-like)是植物特有的转录因子,研究表明其在参与发育阶段转变、花和果实发育等方面起着重要作用。利用PCR技术从白桦基因组DNA中扩增获得BpSPL2基因上游1 960 bp启动子序列,使用PLACE和Plant CARE在线软件分析序列,发现BpSPL2基因启动子序列中含有与开花、非生物胁迫及激素响应等相关的顺式作用元件,暗示其在植物的生长发育和胁迫应答中起重要作用。进而构建了BpSPL2基因启动子驱动GUS报告基因的植物表达载体,并利用农杆菌介导将其瞬时转化至白桦和拟南芥,通过GUS组织化学染色检测BpSPL2基因启动子的组织表达特性,结果表明BpSPL2基因启动子具有启动子活性,能够驱动GUS基因在白桦和拟南芥中表达;而其表达活性在白桦的叶片、芽及根部中较强,在拟南芥的花药、雌蕊和叶片较强,为进一步研究白桦BpSPL2基因的表达调控及其功能分析提供参考。     

桃PpMADS1基因启动子的克隆及功能分析

PpMADS1基因属于一类MADS box 基因,在植物的花发育调控中起着重要的作用。通过Genome Walking的方法从桃基因组中分离了长度为1 814bp的PpMADS1基因启动子片段,序列分析表明,在此启动子上不仅含有TATA box 和CAAT box基本元件,而且含有大量的与光调节有关的调控元件,如GT-1,Sp1和as-2-box,另外存在两个CArG-box元件、一个G-box元件和一个TGA-element,说明该启动子可能受光周期和激素的调控。将该启动子通过5′端缺失,分区段与GUS报告基因连接构建表达载体,并转化拟南芥。GUS组织化学染色分析结果表明,在-197到-454bp有促使GUS在花原基中表达的花原基特异性元件,在-454到-678bp之间存在促使GUS在萼片和花瓣表达的特异性元件,在-678到-978bp存在负调控作用元件,阻遏了GUS基因在花药中的表达。     

拟南芥FRUITFULL(FUL)基因的表达调控模式

FRUITFULL(FUL)基因是一类MADS box基因,在控制开花时间、花分生组织分化、茎生叶形态以及心皮和果实的发育中发挥重要作用。为了阐明FUL的表达调控模式,克隆了拟南芥Arabidopsis thaliana FUL启动子区(-2148bp~+96bp)及其第一内含子,并构建一系列启动子分段缺失表达载体及含FUL第一内含子的融合载体。并进一步构建了各顺式作用元件融合拟南芥TUBULIN和ACTIN启动子的表达载体。转基因拟南芥分析结果表明,FUL启动子的上游存在2个抑制其表达的顺式作用元件,其中一个很可能与转录因子AP1的结合有关;2个存在于上游调控区的CArG-box对FUL基因表达起到重要的调控作用;FUL基因第一内含子参与拟南芥心皮和雄蕊的发育调控,而且有增强基因表达的作用。     

拟南芥GHMP基因家族成员的组织表达及生物信息学分析

利用生物信息学方法获得拟南芥全基因组中12个GHMP基因家族成员。通过实时定量PCR技术研究这12个基因在不同组织中的表达,结果显示它们具有组织表达特异性。构建了拟南芥中GHMP基因家族成员的系统进化树。启动子区调控元件分析表明,大多数GHMP成员包含有光响应、生物钟及其它逆境胁迫响应的相关元件,预测这些GHMP基因家族成员可能参与了植物的光信号、生物钟及相关的逆境胁迫信号转导途径。     

拟南芥株型突变体zpr1的性状特征与基因鉴定分析

对本研究室经T-DNA插入法获得的拟南芥株型突变株系——隐性突变体zpr1植株进行植物学性状调查和遗传分析,并对该突变基因进行鉴定、表达定位和调控元件分析。结果显示:(1)性状分析表明,与野生型拟南芥Ws-2相比,突变体zpr1的茎生叶分枝数量增加,茎生叶分枝发生于拟南芥顶端花序部位;野生型拟南芥茎生叶为披针形,而突变体zpr1没有出现分枝的茎生叶呈倒卵形,出现分枝的茎生叶呈披针型;突变体zpr1的主花序高度、株高、分枝高度和分枝长度都高于野生型,且分枝数多于野生型。(2)利用质粒挽救和反向PCR法(IPCR)确定了ZPR1基因突变发生位置是该基因起始密码子上游426bp处,证明T-DNA插入破坏了ZPR1基因的启动子区域,导致该基因在拟南芥内不能正常表达。(3)基因转录调控区域的顺式作用元件分析发现在ZPR1基因的转录调控区有多个与植物激素相关的调控元件,还有与光周期调节相关的调控元件。(4)亚细胞定位发现,ZPR1基因在所有细胞中的细胞膜中表达,而在部分细胞的细胞膜、细胞质和细胞核中均有表达。研究表明,ZPR1基因的表达对植物株型发育有重要的调控作用,该基因的表达水平受植物激素和光照的调节,最终导致了植物株型的变化。     

玉米脱氧麦根酸分泌通道蛋白基因YS3启动子的克隆与启动活性分析

缺铁是世界范围内农业生产面临的严重问题,玉米通过分泌脱氧麦根酸(2’-deoxymugineic acid, DMA)吸收利用土壤中的难溶性铁。为探明玉米DMA分泌通道蛋白基因YS3的表达和调控机制,本文通过克隆获得长为2813 bp的YS3基因启动子,该序列含有大量TATA-box、CAAT-box等启动子基本元件,以及光响应、激素调控等多个顺式调控元件;构建YS3启动子驱动GUS基因的植物表达重组载体pCAMBIA-YS3GUS,利用农杆菌介导转化拟南芥,获得pYS3::GUS转基因植株,对转基因植株进行GUS组织化学染色,并通过石蜡切片技术对转基因植株进行组织观察,分析pYS3::GUS转基因植株中YS3基因启动子的活性。结果表明,YS3启动子主要驱动GUS基因在拟南芥根部表达,且主要集中在根部表皮细胞,机械损伤可激发YS3启动子活性,驱动GUS基因在损伤临近部位表达。本研究对于理解玉米DMA分泌的分子调控机理方法od3 gmaigensuan有重要意义。     

拟南芥甲基结合蛋白基因AtMBP11启动子在种子形成和萌发中的功能鉴定

为研究拟南芥甲基结合蛋白基因AtMBP11在种子形成和萌发过程中的调控模式,克隆拟南芥AtMBP11启动子,将其替换植物表达载体pBI121的35S启动子序列,转入拟南芥基因组中.转基因拟南芥后代卡那霉素抗性发生分离,选取具有3∶1分离比的后代自交,产生纯合的具有单拷贝插入的后代.转基因后代GUS染色结果表明,新克隆的MBP启动子控制基因在种子、花药和花粉中高效表达.通过对AtMBP11核心启动子缺失分析表明,G-box元件是主要功能元件.     

油棕DGAT2基因启动子克隆及表达的组织特异性研究

以油棕(Elaeis guineensis Jacq.)叶片基因组DNA为模板,克隆获得长度为1035 bp的二酰甘油酰基转移酶基因(DGAT2)的启动子区序列。序列分析结果表明,DGAT2基因启动子含有大量光反应元件、激素响应元件及部分转录因子结合位点。本研究同时构建了DGAT2基因启动子和GUS基因植物融合表达载体,通过蘸花法侵染拟南芥(Arabidopsis thaliana L.),并对转基因拟南芥中GUS基因表达的特异性进行了分析。结果显示,GUS基因在拟南芥各组织中均有表达,但没有明显的组织特异性;荧光定量PCR分析结果表明DGAT2在油棕不同器官中的转录水平存在明显差异。     

大豆Glyma03g24460基因功能预测及表达分析

大豆Glyma03g24460基因,与拟南芥ECERIFERUM1(CER1)基因具有高度的同源性,是一种植物角质层蜡质基因,参与植物角质层蜡质的合成。本研究对其进行基因功能预测和表达分析,利用Plant CARE分析启动子元件发现在基因启动子序列中含有黄酮合成、激素响应、生物和非生物胁迫相关的元件。氨基酸序列比对发现Glyma03g24460与其他物种的脂肪醛脱羧酶基因家族成员有很高的相似度。Glyma03g24460在大豆的q RT-PCR结果表明,它主要在植物的地上器官表达,并且可以受到ABA及干旱等非生物胁迫的诱导表达。     

Plant sulfite oxidase as novel producer of H2O2: combination of enzyme catalysis with a subsequent non-enzymatic reaction step

Sulfite oxidase (EC 1.8.3.1) from the plant Arabidopsis thaliana is the smallest eukaryotic molybdenum enzyme consisting of a molybdenum cofactor-binding domain but lacking the heme domain that is known from vertebrate sulfite oxidase. While vertebrate sulfite oxidase is a mitochondrial enzyme with cytochrome c as the physiological electron acceptor, plant sulfite oxidase is localized in peroxisomes and does not react with cytochrome c. Here we describe results that identified oxygen as the terminal electron acceptor for plant sulfite oxidase and hydrogen peroxide as the product of this reaction in addition to sulfate. The latter finding might explain the peroxisomal localization of plant sulfite oxidase. 18O labeling experiments and the use of catalase provided evidence that plant sulfite oxidase combines its catalytic reaction with a subsequent non-enzymatic step where its reaction product hydrogen peroxide oxidizes another molecule of sulfite. In vitro, for each catalytic cycle plant SO will bring about the oxidation of two molecules of sulfite by one molecule of oxygen. In the plant, sulfite oxidase could be responsible for removing sulfite as a toxic metabolite, which might represent a means to protect the cell against excess of sulfite derived from SO2 gas in the atmosphere (acid rain) or during the decomposition of sulfur-containing amino acids. Finally we present a model for the metabolic interaction between sulfite and catalase in the peroxisome.     

Significance of plant sulfite oxidase

Sulfite oxidizing activities are known since years in animals, microorganisms, and also plants. Among plants, the only enzyme well characterized on molecular and biochemical level is the molybdoenzyme sulfite oxidase (SO). It oxidizes sulfite using molecular oxygen as electron acceptor, leading to the production of sulfate and hydrogen peroxide. The latter reaction product seems to be the reason why plant SO is localized in peroxisomes, because peroxisomal catalase is able to decompose hydrogen peroxide. On the other hand, we have indications for an additional reaction taking place in peroxisomes: sulfite can be nonenzymatically oxidized by hydrogen peroxide. This will promote the detoxification of hydrogen peroxide especially in the case of high amounts of sulfite. Hence we assume that SO could possibly serve as "safety valve" for detoxifying excess amounts of sulfite and protecting the cell from sulfitolysis. Supportive evidence for this assumption comes from experiments where we fumigated transgenic poplar plants overexpressing ARABIDOPSIS SO with SO(2) gas. In this paper, we try to explain sulfite oxidation in its co-regulation with sulfate assimilation and summarize other sulfite oxidizing activities described in plants. Finally we discuss the importance of sulfite detoxification in plants.     

Identification and biochemical characterization of Arabidopsis thaliana sulfite oxidase. A new player in plant sulfur metabolism

In mammals and birds, sulfite oxidase (SO) is a homodimeric molybdenum enzyme consisting of an N-terminal heme domain and a C-terminal molybdenum domain (EC ). In plants, the existence of SO has not yet been demonstrated, while sulfite reductase as part of sulfur assimilation is well characterized. Here we report the cloning of a plant sulfite oxidase gene from Arabidopsis thaliana and the biochemical characterization of the encoded protein (At-SO). At-SO is a molybdenum enzyme with molybdopterin as an organic component of the molybdenum cofactor. In contrast to homologous animal enzymes, At-SO lacks the heme domain, which is evident both from the amino acid sequence and from its enzymological and spectral properties. Thus, among eukaryotes, At-SO is the only molybdenum enzyme yet described possessing no redox-active centers other than the molybdenum. UV-visible and EPR spectra as well as apparent K(m) values are presented and compared with the hepatic enzyme. Subcellular analysis of crude cell extracts showed that SO was mostly found in the peroxisomal fraction. In molybdenum cofactor mutants, the activity of SO was strongly reduced. Using antibodies directed against At-SO, we show that a cross-reacting protein of similar size occurs in a wide range of plant species, including both herbacious and woody plants.     

Sulfite oxidizing enzymes

Sulfite oxidizing enzymes are essential mononuclear molybdenum (Mo) proteins involved in sulfur metabolism of animals, plants and bacteria. There are three such enzymes presently known: (1) sulfite oxidase (SO) in animals, (2) SO in plants, and (3) sulfite dehydrogenase (SDH) in bacteria. X-ray crystal structures of enzymes from all three sources (chicken SO, Arabidopsis thaliana SO, and Starkeya novella SDH) show nearly identical square pyramidal coordination around the Mo atom, even though the overall structures of the proteins and the presence of additional cofactors vary. This structural information provides a molecular basis for studying the role of specific amino acids in catalysis. Animal SO catalyzes the final step in the degradation of sulfur-containing amino acids and is critical in detoxifying excess sulfite. Human SO deficiency is a fatal genetic disorder that leads to early death, and impaired SO activity is implicated in sulfite neurotoxicity. Animal SO and bacterial SDH contain both Mo and heme domains, whereas plant SO only has the Mo domain. Intraprotein electron transfer (IET) between the Mo and Fe centers in animal SO and bacterial SDH is a key step in the catalysis, which can be studied by laser flash photolysis in the presence of deazariboflavin. IET studies on animal SO and bacterial SDH clearly demonstrate the similarities and differences between these two types of sulfite oxidizing enzymes. Conformational change is involved in the IET of animal SO, in which electrostatic interactions may play a major role in guiding the docking of the heme domain to the Mo domain prior to electron transfer. In contrast, IET measurements for SDH demonstrate that IET occurs directly through the protein medium, which is distinctly different from that in animal SO. Point mutations in human SO can result in significantly impaired IET or no IET, thus rationalizing their fatal effects. The recent developments in our understanding of sulfite oxidizing enzyme mechanisms that are driven by a combination of molecular biology, rapid kinetics, pulsed electron paramagnetic resonance (EPR), and computational techniques are the subject of this review.     

Cloning of an ovule specific promoter from Arabidopsis thaliana and expression of beta-glucuronidase

Tissue specific expression of transgenes in plant species has several advantages over constitutive expression. Identification of ovule specific promoters would be useful in genetic engineering of plants with a variety of desirable traits such as genetically engineered parthenocarpy, female sterile plants or seedless fruits. Relative inaccessibility and difficulty in harvesting adequate amounts of tissue at known developmental stages has impeded the progress in cloning of promoters involved in ovule development. In the present study an ovule specific promoter was cloned from Arabidopsis AGL11 gene and used to express GUS (beta-glucuronidase) gene in transgenic Arabidopsis. Histochemical staining of GUS appeared in the center of young ovary (ovules), but no detectable GUS activity was observed in vegetative plant tissues, sepals, petals and androecium. AGL11 gene promoter can be useful to modify the developmental path of plants by expressing either plant hormones or lethal genes for agronomic purpose.     

Peroxisomal localization of sulfite oxidase separates it from chloroplast-based sulfur assimilation

Recently, we isolated the sulfite oxidase (SO) gene from Arabidopsis thaliana and characterized the purified SO protein. The purpose of the present study was to determine the subcellular localization of this novel plant enzyme. Immunogold electron-microscopic analysis showed the gold labels nearly exclusively in the peroxisomes. To verify this finding, green fluorescent protein was fused to full-length plant SO including the putative peroxisomal targeting signal 1 (PTS1) 'SNL' and expressed in tobacco leaves. Our results showed a punctate fluorescence pattern resembling that of peroxisomes. Co-labelling with MitoTracker-Red excluded that the observed fluorescence was due to mitochondrial sorting. By investigation of deleted or mutated PTS1, no functional peroxisomal targeting signal 2 (PTS2) could be detected in plant SO. This conclusion is supported by expression studies in Pichia pastoris mutants with defined defects either in PTS1- or PTS2-mediated peroxisomal import.     

Sulfite oxidase controls sulfur metabolism under SO2 exposure in Arabidopsis thaliana

In the present study, the significance of sulfite oxidase (SO) for sulfite detoxification and sulfur assimilation was investigated. In response to sulfur dioxide (SO(2)) exposure, a remarkable expansion of sulfate and a significant increase of GSH pool were observed in wild-type and SO-overexpressing Arabidopsis. These metabolic changes were connected with a negative feedback inhibition of adenosine 5'-phosphosulfate reductase (APR), but no alterations in gas exchange parameters or visible symptoms of injury. However, Arabidopsis SO-KO mutants were consistently negatively affected upon 600 nL L(-1) SO(2) exposure for 60 h and showed phenotypical symptoms of injury with small necrotic spots on the leaves. The mean g(H2O) was reduced by about 60% over the fumigation period, accompanied by a reduction of net CO(2) assimilation and SO(2) uptake of about 50 and 35%. Moreover, sulfur metabolism was completely distorted. Whereas sulfate pool was kept constant, thiol-levels strongly increased. This demonstrates that SO should be the only protagonist for back-oxidizing and detoxification of sulfite. Based on these results, it is suggested that co-regulation of SO and APR controls sulfate assimilation pathway and stabilizes sulfite distribution into organic sulfur compounds. In conclusion, a sulfate-sulfite cycle driven by APR and SO can be postulated for fine-tuning of sulfur distribution that is additionally used for sulfite detoxification, when plants are exposed to atmospheric SO(2).