1-脱氧木酮糖-5-磷酸合成酶(DXS)及其编码基因

萜类物质是广泛分布于生物界的一类天然产物,也是重要生命物质。萜类物质通过甲羟戊酸(MVA)途径和2-C-甲基-D-赤藻糖醇-4-磷酸(MEP)途径合成,古细菌、真菌和动物及人的萜类物质主要通过MVA途径合成,而多数真细菌(即通常而言的细菌)则利用MEP途径。植物同时拥有两种途径但分别定位于细胞质和质体。1-脱氧木酮糖-5-磷酸合成酶(DXS)是MEP途径的第一个酶,也是该途径的关键调控位点。现从DXS在MEP途径中的作用、DXS结构、亚细胞定位和酶活性、编码基因及突变体等方面对DXS进行全面阐述。拟南芥DXS基因插入突变体cla1-1发生白化,DXS基因表达与类胡萝卜素等萜类物质积累密切相关,在转基因生物体中过度表达DXS可促进萜类物质合成。植物DXS具有典型的质体转运肽序列,决定了DXS的质体定位。完备的DXS活性分析体系为DXS抑制剂开发筛选等研究奠定良好基础。DXS由一至多个基因编码,随生物种类而异,根据同源性,植物DXS基因可分成两类。DXS基因家族不同成员具有不同的表达模式,但通常有一个成员在多种组织中广泛表达。     

Isolation and characterization of root-specific phosphate transporter promoters from Medicago trunatula

Promoters of phosphate transporter genes MtPT1 and MtPT2 of Medicago truncatula were isolated by utilizing the gene-space sequence information and by screening of a genomic library, respectively. Two reporter genes, beta-glucuronidase (GUS) and green fluorescent protein (GFP) were placed under the control of the MtPT1 and MtPT2 promoters. These chimeric transgenes were introduced into Arabidopsis thaliana and transgenic roots of M. truncatula, and expression patterns of the reporter genes were assayed in plants grown under different phosphate (Pi) concentrations. The expression of GUS and GFP was only observed in root tissues, and the levels of expression decreased with increasing concentrations of Pi. GUS activities in roots of transgenic plants decreased 10-fold when the plants were transferred from 10 microM to 2 mM Pi conditions, however, when the plants were transferred back to 10 microM Pi conditions, GUS expression reversed back to the original level. The two promoters lead to different expression patterns inside root tissues. The MtPT1 promoter leads to preferential expression in root epidermal and cortex cells, while MtPT2 promoter results in strong expression in the vascular cylinder in the center of roots. Promoter deletion analyses revealed possible sequences involved in root specificity and Pi responsiveness. The promoters are valuable tools for defined engineering of plants, particularly for root-specific expression of transgenes.     

油棕等热带植物DXS基因的生物信息学分析

油棕等热带植物含有丰富的胡萝卜素和维生素E等类异戊二烯物质,类胡萝卜素和甾醇等类异戊二烯物质在植物生命活动中扮演重要角色,并且对保护人类健康具有重要意义,MEP途径是合成类异戊二烯的重要途径之一。DXS是MEP途径中的第一个限速酶,其功能在油棕等热带植物中极其保守。为了弄清油棕等热带植物DXS的结构和功能特点,该研究利用生物信息学工具和软件对以油棕等热带植物类异戊二烯合成关键基因DXS为对象,进行核酸和氨基酸序列的理化性质、蛋白质结构以及功能结构域等分析,探讨了不同物种间的亲缘关系。结果表明:DXS基因起始密码子均为ATG,终止密码子则分为TAG、TAA和TGA,DXS蛋白质属于不具有信号肽的亲水性蛋白,可能作为转运蛋白在叶绿体基质中发挥作用,未发现明显的跨膜结构域,磷酸化位点有36个,其中丝氨酸、苏氨酸和酪氨酸位点分别为17、11和8个,无规则卷曲和α-螺旋是蛋白质二级结构主要的结构元件,三级结构预测具有DXS酶特征,硫胺素焦磷酸盐结合位点和PLN02582保守结构域,不同植物DXS功能结构域非常保守,可以作为判断不同物种间亲缘关系的重要依据。该研究结果为油棕等热带植物DXS的结构、功能分析和利用提供了进一步的信息,为其品质性状分子机制研究及遗传改良奠定了基础。     

Flavone synthases from Medicago truncatula are flavanone-2-hydroxylases and are important for nodulation

Flavones are important copigments found in the flowers of many higher plants and play a variety of roles in plant adaptation to stress. In Medicago species, flavones also act as signal molecules during symbiotic interaction with the diazotropic bacterium Sinorhizobium meliloti. They are the most potent nod gene inducers found in root exudates. However, flavone synthase II (FNS II), the key enzyme responsible for flavone biosynthesis, has not been characterized in Medicago species. We cloned two FNS II genes from Medicago truncatula using known FNS II sequences from other species and named them MtFNSII-1 and MtFNSII-2. Functional assays in yeast (Saccharomyces cerevisiae) suggested that the catalytic mechanisms of both cytochrome P450 monooxygenases were similar to the other known legume FNS II from licorice (Glycyrrhiza echinata). MtFNSII converted flavanones to 2-hydroxyflavanones instead of flavones whereas FNS II from the nonlegume Gerbera hybrida, converted flavanones to flavones directly. The two MtFNSII genes had distinct tissue-specific expression patterns. MtFNSII-1 was highly expressed in roots and seeds whereas MtFNSII-2 was highly expressed in flowers and siliques. In addition, MtFNSII-2 was inducible by S. meliloti and methyl jasmonate treatment, whereas MtFNSII-1 was not. Histochemical staining of transgenic hairy roots carrying the promoter-reporter constructs indicated that the MtFNSII-2 induction was tissue specific, mostly localized to vascular tissues and root hairs. RNA interference-mediated suppression of MtFNSII genes resulted in flavone depleted roots and led to significantly reduced nodulation when inoculated with S. meliloti. Our results provide genetic evidence supporting that flavones are important for nodulation in M. truncatula.     

<Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis>-induced chitinase gene expression in<Emphasis Type="Italic"> Medicago truncatula</Emphasis> ecotype R108-1: a comparison between symbiosis-specific class V and defence-related class IV chitinases

The Medicago truncatula (Gaertn.) ecotypes Jemalong A17 and R108-1 differ in Sinorhizobium meliloti-induced chitinase gene expression. The pathogen-inducible class IV chitinase gene, Mtchit 4, was strongly induced during nodule formation of the ecotype Jemalong A17 with the S. meliloti wild-type strain 1021. In the ecotype R108-1, the S. meliloti wild types Sm1021 and Sm41 did not induce Mtchit 4 expression. On the other hand, expression of the putative class V chitinase gene, Mtchit 5, was found in roots of M. truncatula cv. R108-1 nodulated with either of the rhizobial strains. Mtchit 5 expression was specific for interactions with rhizobia. It was not induced in response to fungal pathogen attack, and not induced in roots colonized with arbuscular mycorrhizal (AM) fungi. Elevated Mtchit 5 gene expression was first detectable in roots forming nodule primordia. In contrast to Mtchit 4, expression of Mtchit 5 was stimulated by purified Nod factors. Conversely, Mtchit 4 expression was strongly elevated in nodules formed with the K-antigen-deficient mutant PP699. Expression levels of Mtchit 5 were similarly increased in nodules formed with PP699 and its parental wild-type strain Sm41. Phylogenetic analysis of the deduced amino acid sequences of Mtchit 5 (calculated molecular weight = 41,810 Da, isoelectric point pH 7.7) and Mtchit 4 (calculated molecular weight 30,527 Da, isoelectric point pH 4.9) revealed that the putative Mtchit 5 chitinase forms a separate clade within class V chitinases of plants, whereas the Mtchit 4 chitinase clusters with pathogen-induced class IV chitinases from other plants. These findings demonstrate that: (i) Rhizobium-induced chitinase gene expression in M. truncatula occurs in a plant ecotype-specific manner, (ii) Mtchit 5 is a putative chitinase gene that is specifically induced by rhizobia, and (iii) rhizobia-specific and defence-related chitinase genes are differentially influenced by rhizobial Nod factors and K antigens.     

The Medicago truncatula lysin [corrected] motif-receptor-like kinase gene family includes NFP and new nodule-expressed genes

Rhizobial Nod factors are key symbiotic signals responsible for starting the nodulation process in host legume plants. Of the six Medicago truncatula genes controlling a Nod factor signaling pathway, Nod Factor Perception (NFP) was reported as a candidate Nod factor receptor gene. Here, we provide further evidence for this by showing that NFP is a lysin [corrected] motif (LysM)-receptor-like kinase (RLK). NFP was shown both to be expressed in association with infection thread development and to be involved in the infection process. Consistent with deviations from conserved kinase domain sequences, NFP did not show autophosphorylation activity, suggesting that NFP needs to associate with an active kinase or has unusual functional characteristics different from classical kinases. Identification of nine new M. truncatula LysM-RLK genes revealed a larger family than in the nonlegumes Arabidopsis (Arabidopsis thaliana) or rice (Oryza sativa) of at least 17 members that can be divided into three subfamilies. Three LysM domains could be structurally predicted for all M. truncatula LysM-RLK proteins, whereas one subfamily, which includes NFP, was characterized by deviations from conserved kinase sequences. Most of the newly identified genes were found to be expressed in roots and nodules, suggesting this class of receptors may be more extensively involved in nodulation than was previously known.     

利用类萜代谢工程改良作物风味

类萜是从植物中分离出的一类类异戊二烯物质。其中挥发性萜类除了在吸引授粉媒、异株克生和植物防御中起到一定的生态作用外,还影响到水果、蔬菜和其他作物的香味形成。对类萜生物合成及其代谢工程的最新研究进展进行了综述,探讨了代谢过程中的关键酶基因,尤其是类萜合成酶(TPSs)基因的表达特性以及操纵类萜生物合成途径提高产量的几种可能的策略。随着更多相关基因的分离,利用代谢工程人工改良作物风味将指日可待。     

Identification and characterization of class 1 <Emphasis Type="Italic">DXS</Emphasis> gene encoding 1-deoxy-<Emphasis Type="SmallCaps">d</Emphasis>-xylulose-5-phosphate synthase,the first committed enzyme of the MEP pathway from soybean

1-Deoxy-d-xylulose-5-phosphate synthase (DXS) catalyses the first committed step of the 2C-methyl-d-erythritol-4-phosphate (MEP) pathway, which is an alternative isoprenoids biosynthetic route that has been recently discovered. In this work, a DXS1-like cDNA (GmDXS1) was isolated from soybean. The full-length cDNA of GmDXS1 encoded 708 amino acid residues with a predicted molecular mass of 76.4 KD. Sequence alignment showed that GmDXS1 had high homology to known DXS proteins from other plant species and contained the conserved N-terminal plastid transit peptide, the N-terminal thiamine binding domain and pyridine binding DRAG domain. Phylogenetic analysis indicated that GmDXS1 belonged to the plant DXS1 cluster. Southern blot analysis indicated that a single copy of GmDXS1 gene existed in soybean genome. Tissue expression analysis revealed that GmDXS1 expressed in all photosynthetic tissues except pod walls and roots. Green fluorescence analysis with the fusion protein 35S:GmDXS1:GFP suggested that GmDXS1 was localized in plastid. The relatively higher photosynthetic pigment content in transgenic tobacco leaves compared to the control implied that GmDXS1 catalyzed the first potential regulatory step in photosynthetic pigment biosynthesis via the MEP pathway.     

Apocarotenoid biosynthesis in arbuscular mycorrhizal roots: contributions from methylerythritol phosphate pathway isogenes and tools for its manipulation

During colonization by arbuscular mycorrhizal (AM) fungi plant roots frequently accumulate two types of apocarotenoids (carotenoid cleavage products). Both compounds, C(14) mycorradicin and C(13) cyclohexenone derivatives, are predicted to originate from a common C(40) carotenoid precursor. Mycorradicin is the chromophore of the "yellow pigment" responsible for the long-known yellow discoloration of colonized roots. The biosynthesis of apocarotenoids has been investigated with a focus on the two first steps of the methylerythritol phosphate (MEP) pathway catalyzed by 1-deoxy-D-xylulose 5-phosphate synthase (DXS) and 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR). In Medicago truncatula and other plants the DXS2 isogene appears to be specifically involved in the AM-mediated accumulation of apocarotenoids, whereas in the case of DXR a single gene contributes to both housekeeping and mycorrhizal (apo)carotenoid biosynthesis. Immunolocalization of DXR in mycorrhizal maize roots indicated an arbuscule-associated protein deposition, which occurs late in arbuscule development and accompanies arbuscule degeneration and breakdown. The DXS2 isogene is being developed as a tool to knock-down apocarotenoid biosynthesis in mycorrhizal roots by an RNAi strategy. Preliminary results from this approach provide starting points to suggest a new kind of function for apocarotenoids in mycorrhizal roots.     

Enhanced flux through the methylerythritol 4-phosphate pathway in Arabidopsis plants overexpressing deoxyxylulose 5-phosphate reductoisomerase

The methylerythritol 4-phosphate (MEP) pathway synthesizes the precursors for an astonishing diversity of plastid isoprenoids, including the major photosynthetic pigments chlorophylls and carotenoids. Since the identification of the first two enzymes of the pathway, deoxyxylulose 5-phoshate (DXP) synthase (DXS) and DXP reductoisomerase (DXR), they both were proposed as potential control points. Increased DXS activity has been shown to up-regulate the production of plastid isoprenoids in all systems tested, but the relative contribution of DXR to the supply of isoprenoid precursors is less clear. In this work, we have generated transgenic Arabidopsis thaliana plants with altered DXS and DXR enzyme levels, as estimated from their resistance to clomazone and fosmidomycin, respectively. The down-regulation of DXR resulted in variegation, reduced pigmentation and defects in chloroplast development, whereas DXR-overexpressing lines showed an increased accumulation of MEP- derived plastid isoprenoids such as chlorophylls, carotenoids, and taxadiene in transgenic plants engineered to produce this non-native isoprenoid. Changes in DXR levels in transgenic plants did not result in changes in␣DXS gene expression or enzyme accumulation, confirming that the observed effects on plastid isoprenoid levels in DXR-overexpressing lines were not an indirect consequence of altering DXS levels. The results indicate that the biosynthesis of MEP (the first committed intermediate of the pathway) limits the production of downstream isoprenoids in Arabidopsis chloroplasts, supporting a role for DXR in the control of the metabolic flux through the MEP pathway.     

Response of <Emphasis Type="Italic">Pisum Sativum</Emphasis> Cytokinin Oxidase/Dehydrogenase Expression and Specific Activity to Drought Stress and Herbicide Treatments

The expression of cytokinin oxidase/dehydrogenase (CKX EC: 1.5.99.12) is subject to fine regulation and it provides a rapid turnover of cytokinins, which serves as a signal for triggering developmental events during plant growth. The activity of this enzyme is believed to be responsible for the changes in cytokinin pool under adverse environmental conditions. CKX gene-specific assay to measure the expression in response to different stress treatments in the tissues of Pisum sativum plants was developed. Pea CKX genes were amplified and sequenced using primers designed from the sequences of Medicago truncatula CKX genes. Expression of two P. sativum CKX genes was assessed using relative-quantification in real time two-step RT-PCR, in leaves and roots of drought-, glufosinate- and atrazine-treated cv. Manuela pea plants. Varied CKX responses support the existence of complicated regulating mechanism of cytokinin oxidase/ dehydrogenase gene expression.     

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.