Cell Specific,Cross-Species Expression of Myrosinases in Brassica Napus,Arabidopsis Thaliana and Nicotiana Tabacum

A prototypical characteristic of the Brassicaceae is the presence of the myrosinase-glucosinolate system. Myrosinase, the only known S-glycosidase in plants, degrades glucosinolates, thereby initiating the formation of isothiocyanates, nitriles and other reactive products with biological activities. We have used myrosinase gene promoters from Brassica napus and Arabidopsis thaliana fused to the beta -glucuronidase (GUS) reporter gene and introduced into Arabidopsis thaliana, Brassica napus and/or Nicotiana tabacum plants to compare and determine the cell types expressing the myrosinase genes and the GUS expression regulated by these promoters. The A. thaliana TGG1 promoter directs expression to guard cells and phloem myrosin cell idioblasts of transgenic A. thaliana plants. Expression from the same promoter construct in transgenic tobacco plants lacking the myrosinase enzyme system also directs expression to guard cells. The B. napus Myr1.Bn1 promoter directs a cell specific expression to idioblast myrosin cells of immature and mature seeds and myrosin cells of phloem of B. napus. In A. thaliana the B. napus promoter directs expression to guard cells similar to the expression pattern of TGG1. The Myr1.Bn1 signal peptide targets the gene product to the reticular myrosin grains of myrosin cells. Our results indicate that myrosinase gene promoters from Brassicaceae direct cell, organ and developmental specific expression in B. napus, A. thaliana and N. tabacum.     

A Brassica S locus gene promoter directs sporophytic expression in the anther tapetum of transgenic Arabidopsis

The S locus glycoprotein (SLG) gene of Brassica encodes stigmatic glycoproteins that are implicated in the pollen-stigma interaction of self-incompatibility. We have transformed the related plant Arabidopsis thaliana with a chimaeric gene consisting of the promoter region of an SLG gene fused to the reporter gene beta-glucuronidase (GUS). In transgenic plants the gene was expressed in two cell types of the flower. In stigmas, the timing and distribution of GUS activity was similar to that previously described for SLG expression in Brassica. In anthers, expression was detected at an earlier stage of flower development with GUS activity restricted to the tapetal cell layer. The novel finding of SLG-promoter activity in the anther supports the hypothesis that sporophytic control of self-incompatibility is a result of SLG-gene expression in the tapetum.     

Regulation of BN115, a low-temperature-responsive gene from winter Brassica napus.

The genomic clone for BN115, a low-temperature-responsive gene, was isolated from winter Brassica napus and its sequence was determined. A 1.2-kb fragment of the 5' regulatory region (from bp -1107 to +100) was fused to the beta-glucuronidase (GUS) reporter gene and BN115-promoted GUS expression was observed in green tissues of transgenic B. napus plants only after incubation at 2 degrees C. No expression was observed after incubation at 22 degrees C, either in the presence or the absence of ABA. Microprojectile bombardment of winter B. napus leaves with a BN115 promoter/GUS construct yielded similar results and was used to analyze a series of deletions from the 5' end of the promoter. Results obtained from transient expression studies showed that the low-temperature regulation of BN115 expression involves a possible enhancer region between bp -1107 and -802 and a second positive regulatory region located between bp -302 and -274. Deletion analyses and results from replacement with a truncated cauliflower mosaic virus 35S promoter suggest that the minimal size required for any maintenance of low-temperature GUS expression is a -300-bp fragment. Within this fragment are two 8-bp elements with the sequence TGGCCGAC, which are identical to those present in the positive regulatory region of the promoter of the homologous Arabidopsis cor15a gene and to a 5-bp core sequence in the low-temperature- and dehydration-responsive elements identified in the promoter regions of several cold-responsive Arabidopsis thaliana genes.     

Antisense inhibition of flavonoid biosynthesis in petunia anthers results in male sterility.

Inhibition of flower pigmentation in transgenic petunia plants was previously accomplished by expressing an antisense chalcone synthase (chs) gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter. This chimeric gene was not effective in inhibiting pigmentation in anthers, presumably because the viral CaMV 35S promoter was insufficiently expressed in cell types of this organ in which the pigments are produced. Insertion of the anther box, a homologous sequence found in other genes expressed in anthers, resulted in a modified expression pattern driven by this promoter, as monitored by the beta-glucuronidase (gus) gene. In addition to the basic CaMV 35S expression pattern in anthers, GUS activity was observed in tapetum cells when the modified promoter was fused to the gus gene. This promoter construct was subsequently used to drive an antisense chs gene in transgenic petunia, which led to the inhibition of pigment synthesis in anthers of five of 35 transformants. Transgenic plants with white anthers were male sterile due to an arrest in male gametophyte development. This finding indicated that flavonoids play an essential role in male gametophyte development.     

Expression patterns of duplicate tryptophan synthase beta genes in Arabidopsis thaliana.

Expression of the two Arabidopsis thaliana genes encoding tryptophan synthase beta (TSB1 and TSB2) was investigated by gene-specific RNA blot hybridization and reporter gene analysis. TSB1 mRNA abundance varies in an organ-specific manner, whereas TSB2 mRNA does not. Quantitative analysis of transgenic plants expressing TSB1 and TSB2 translational fusions to the beta-glucuronidase (GUS) gene (gusA) indicates that TSB1-GUS activity is 15-fold higher than TSB2-GUS. Histochemical analysis of these transgenic A. thaliana plants indicates that GUS expression occurs in a developmentally regulated manner. GUS activity driven from the TSB1 promoter is predominantly associated with the stem, root tips, foliar vasculature, mesophyll cells, base of developing seed pods, and tips of anther filaments in plants 15 d and older. Sections through the vegetative stem reveal GUS staining in all cell types including the shoot apical meristem. Although TSB2-GUS expression is consistently detected in root tips and at the base of developing seed pods, it is observed later in plant development than is TSB1-GUS expression.     

Pollen-specific gene expression in transgenic plants: coordinate regulation of two different tomato gene promoters during microsporogenesis

To investigate the regulation of gene expression during male gametophyte development, we analyzed the promoter activity of two different genes (LAT52 and LAT59) from tomato, isolated on the basis of their anther-specific expression. In transgenic tomato, tobacco and Arabidopsis plants containing the LAT52 promoter region fused to the beta-glucuronidase (GUS) gene, GUS activity was restricted to pollen. Transgenic tomato, tobacco and Arabidopsis plants containing the LAT59 promoter region fused to GUS also showed very high levels of GUS activity in pollen. However, low levels of expression of the LAT59 promoter construct were also detected in seeds and roots. With both constructs, the appearance of GUS activity in developing anthers was correlated with the onset of microspore mitosis and increased progressively until anthesis (pollen shed). Our results demonstrate co-ordinate regulation of the LAT52 and LAT59 promoters in developing microspores and suggest that the mechanisms that regulate pollen-specific gene expression are evolutionarily conserved.     

A promoter directing high level expression in pistils of transgenic plants

The promoter of the potato (Solanum tuberosum L.) SK2 gene, encoding a pistil-specific basic endochitinase, was cloned. Various fragments of the SK2-promoter, from 1 kb down to 0.23 kb in length, were fused to the GUS reporter gene. Chimaeric SK2 promoter-GUS fusion constructs were transformed into potato by Agrobacterium tumefaciens-mediated transformation. The SK2-GUS transgenic potato plants exhibited a highly specific GUS activity in the pistil. Expression in the pistil was shown to be developmentally regulated. In addition to the GUS activity in pistils, transgenic plants also showed a much weaker ectopic expression in anthers. In other tissues no systematic expression was detectable. All SK2 promoter fragments analysed conferred pistil-specific expression without significant qualitative or quantitative differences, demonstrating that the regulatory elements mediating this expression pattern are located within a 230 bp SK2 promoter fragment. The SK2 promoter may be used to engineer high levels of expression in pistils of transgenic plants.     

AtSTP3启动子控制的外源基因在转基因水稻中的特异表达研究

利用PCR技术从哥伦比亚型拟南芥基因组DNA中分离了AtSTP3绿色组织特异表达的启动子,序列分析表明,扩增片段(1774bp)与已报道序列的相应区域同源性达99.9%。将其与GUS报告基因融合在一起,构建了植物表达载体,并由农杆菌介导法导入水稻品种‘中花11’中。对转基因水稻植株中的GUS活性进行定性与定量测定结果表明,AtSTP3启动子可驱动GUS报告基因在转基因水稻植株叶片中特异性表达,而在根和种子等器官中不表达或表达活性极弱,AtSTP3启动子表现出明显的组织特异性。     

Differential expression of a senescence-enhanced metallothionein gene in Arabidopsis in response to isolates of Peronospora parasitica and Pseudomonas syringae

The metallothionein gene, LSC54 , shows increased expression during leaf senescence in Brassica napus and Arabidopsis thaliana . A number of abiotic and biotic stresses have been shown to induce senescence-like symptoms in plants and, to investigate this further, the promoter of the LSC54 gene was cloned and fused to the GUS gene and transformed into Arabidopsis . The promoter was highly induced during leaf senescence and also in response to wounding; histochemical analysis indicated that this induction was localised to a few cells close to the wound site. The transgenic Arabidopsis tissue was infected with compatible and incompatible isolates of both the fungal biotroph, Peronospora parasitica and the bacterial necrotroph, Pseudomonas syringae. Incompatible isolates induced rapid cell death (the hypersensitive response) at the site of infection and, with both pathogens, early, localised expression of the GUS gene was observed. In contrast, relatively slow induction of the GUS gene was seen in the compatible interaction and this was correlated with the appearance of senescence-like symptoms in the biotrophic interaction and cell death by necrosis that occurred in response to the necrotrophic pathogen. These results suggest that there are common steps in the signalling pathways that lead to cell death in the hypersensitive response, pathogen induced necrosis and senescence.     

拟南芥γ-生育酚甲基转移酶(γ-TMT)启动子的分离及表达特性分析

维生素E是一类人体所必需的脂溶性的维生素,具有重要的生理功能。γ-生育酚甲基转移酶(γ-TMT)是维生素E生物合成途径中的关键酶之一,催化γ、δ-生育酚甲基化,生成α、β-生育酚。从拟南芥中分离了γ-生育酚甲基转移酶基因1552bp的启动子序列,构建了含有该启动子和GUS报告基因的植物表达载体,通过农杆菌介导转化拟南芥,获得了转基因植株。GUS组织化学染色结果表明,在γ-TMT启动子的驱动下,报告基因GUS在拟南芥的叶、茎以及花均有表达,且在茎尖、雄蕊和幼叶中表达最强,而在根、种子和种荚中则没有检测到GUS基因的表达,表明γ-TMT基因可能仅在拟南芥某些组织中特异性高表达。     

A novel promoter from soybean that is active in a complex developmental pattern with and without its proximal 650 base pairs

We report the isolation of a novel soybean gene, Msg, which is highly expressed in developing soybean pods. The gene shows significant homology to a family of fruit- and flower-specific genes, designated the major latex protein (MLP) homologues, so far reported in only a few species and whose functions are unknown. The MLPs are more distantly related to a group of pathogenesis-related proteins (IPR or PR-10) whose functions are likewise unknown. This is the first report of a MLP homologue in a plant for which there is already an IPR-protein reported. We performed an analysis of the Msg promoter with 14 different promoter fragments ranging from 0.65 kb to 2.26 kb, fused to the uidA (GUS) gene. High transient expression was obtained with all the constructs upon particle bombardment in soybean and green bean pods. Stable Arabidopsis transformants were obtained with the Agrobacterium vacuum infiltration method. The promoter is fully active in Arabidopsis only in plants transformed with the 2.26 kb fragment promoter, expressing GUS in nectaries, nodes, short style and in guard cells of the silique, pedicel and stem but not in mature leaves. Surprisingly, the proximal 650 bp TATA-containing region cannot function on its own in Arabidopsis and can be deleted without a change in expression pattern in both Arabidopsis and soybean. Thus, tissue-specific regions of the complex Msg promoter reside in the distal 5 regions upstream of a dispensable TATA box in contrast to many examples of tissue-specific elements that reside much closer to the TATA box.