ASF-2: a factor that binds to the cauliflower mosaic virus 35S promoter and a conserved GATA motif in Cab promoters.

We have used nuclear extracts prepared from tobacco leaf tissue to characterize a factor binding site, designated as-2 (activating sequence-2), at the -100 region of the cauliflower mosaic virus 35S promoter. The activity of this factor, called ASF-2 (activating sequence factor-2), is not detected in tobacco root extracts. as-2 includes two GT motifs with sequence homology to the SV40 enhancer core A element and the Box II element of pea rbcS. Nevertheless, oligomers of these sequence elements do not compete for ASF-2 binding in gel retardation assays, indicating that the GT motifs may not be involved. Methylation interference studies identify two guanines (G93 and G98) that are required for interaction with ASF-2. Sequences surrounding these two critical guanines display homologies to a GATA repeat conserved among several light-responsive promoters. One such sequence from a petunia Cab promoter is able to compete with as-2 for factor binding. In transgenic plants, a tetramer of as-2 is able to confer leaf expression when fused 5' to the -90 derivative of the 35S promoter. The expression is not dependent on light and, thus, the as-2 tetramer does not function as a light-responsive element in this context. Histochemical localization of the reporter gene product suggests that the as-2 tetramer directs expression in trichomes, vascular elements, and epidermal and mesophyll cells.     

Identification of a light-responsive region of the nuclear gene encoding the B subunit of chloroplast glyceraldehyde 3-phosphate dehydrogenase from Arabidopsis thaliana.

We report here the identification of a cis-acting region involved in light regulation of the nuclear gene (GapB) encoding the B subunit of chloroplast glyceraldehyde 3-phosphate dehydrogenase from Arabidopsis thaliana. Our results show that a 664-bp GapB promoter fragment is sufficient to confer light induction and organ-specific expression of the Escherichia coli beta-glucuronidase reporter gene (Gus) in transgenic tobacco (Nicotiana tabacum) plants. Deletion analysis indicates that the -261 to -173 upstream region of the GapB gene is essential for light induction. This region contains four direct repeats with the consensus sequence 5'-ATGAA(A/G)A-3' (Gap boxes). Deletion of all four repeats abolishes light induction completely. In addition, we have linked a 109-bp (-263 to -152) GapB upstream fragment containing the four direct repeats in two orientations to the -92 to +6 upstream sequence of the cauliflower mosaic virus 35S basal promoter. The resulting chimeric promoters are able to confer light induction and to enhance leaf-specific expression of the Gus reporter gene in transgenic tobacco plants. Based on these results we conclude that Gap boxes are essential for light regulation and organ-specific expression of the GapB gene in A. thaliana. Using gel mobility shift assays we have also identified a nuclear factor from tobacco that interacts with GapA and GapB DNA fragments containing these Gap boxes. Competition assays indicate that Gap boxes are the binding sites for this factor. Although this binding activity is present in nuclear extracts from leaves and roots of light-grown or dark-treated tobacco plants, the activity is less abundant in nuclear extracts prepared from leaves of dark-treated plants or from roots of greenhouse-grown plants. In addition, our data show that this binding factor is distinct from the GT-1 factor, which binds to Box II and Box III within the light-responsive element of the RbcS-3A gene of pea.     

Mutation of either G box or I box sequences profoundly affects expression from the Arabidopsis rbcS-1A promoter.

A deletion analysis of the Arabidopsis thaliana rbcS-1A promoter defined a 196 bp region (-320 to -125) sufficient to confer light-regulated expression on a heterologous Arabidopsis alcohol dehydrogenase (Adh) reporter gene in transgenic Nicotiana tabacum (tobacco) leaves. This region, which contains DNA sequences I, G and GT boxes, with homology to other ribulose-1,5-bisphosphate carboxylase small subunit (RBCS) gene promoter sequences, directed expression independent of orientation and relative position in the Adh promoter. Site-specific mutagenesis of these conserved sequences and subsequent expression analysis in transgenic tobacco showed that both G box and I box mutations in the context of the full (-1700 to +21) rbcS-1A promoter substantially reduced the expression of Adh and beta-glucuronidase (GUS) reporter genes. The G box has previously been shown to specifically bind in vitro a factor isolated from nuclear extracts of tomato and Arabidopsis. This factor (GBF) is distinct from the factor GT-1 which binds to adjacent GT boxes in the pea rbcS-3A promoter. Multiple mutations in putative Arabidopsis rbcS-1A promoter GT boxes had no pronounced affect on expression, possibly due to a redundancy of these sites. Experiments in which rbcS-1A promoter fragments were fused to truncated 35S CaMV (cauliflower mosaic virus) promoter--GUS reporter constructs showed that cis-acting CaMV promoter elements could partially restore expression to G-box-mutated rbcS-1A sequences.     

Analysis of regulatory elements involved in the induction of two tobacco genes by salicylate treatment and virus infection.

Tobacco genes encoding the PR-1a protein and a glycine-rich protein are expressed after treatment of plants with salicylate or infection with tobacco mosaic virus. Upstream sequences of these genes were fused to reporter genes, and these constructs were used to transform tobacco. Upstream sequences of the PR-1a gene of 689 base pairs or longer were sufficient for induction of the reporter gene in tobacco mosaic virus-inoculated leaves, systemically induced leaves from infected plants, and leaves treated with salicylate. No such induction was found with upstream sequences of 643 base pairs or shorter of the PR-1a gene. When the PR-1a upstream sequence from nucleotides -625 to -902 was fused to the cauliflower mosaic virus 35S core promoter, a construct was obtained that conferred tobacco mosaic virus and salicylate inducibility to the reporter gene in transgenic plants. This confirmed the localization of tobacco mosaic virus- and salicylate-responsive elements between positions -643 and -689 in the PR-1a promoter. With the glycine-rich protein gene, an upstream sequence of 645 base pairs was sufficient for tobacco mosaic virus and salicylate inducibility of the reporter gene, whereas constructs containing 400 base pairs or fewer of the glycine-rich protein promoter were largely inactive.     

伪狂犬病毒gD基因在转基因烟草中的表达

将猪伪狂犬病毒 (pseudorabiesvirus ,PRV)最主要的保护性抗原基因gD完整编码区亚克隆到修饰的植物双元表达载体pBI 35SL中 ,使其置于强启动子CaMV 35S doubleenhancer TEV 5′UTR下游 ,构建的转基因植物双元表达质粒经农杆菌介导转化烟草 .PCR检测叶片筛选阳性植株 ,Southern杂交进一步证实gD已整合到转基因烟草基因组中 .固相酶联斑点试验和Western印迹表明 ,gD在烟草获得正确表达并具有抗原性     

A/T-rich sequences act as quantitative enhancers of gene expression in transgenic tobacco and potato plants

The role of an A/T-rich positive regulatory region (P268, -444 to -177 from the translation start site) of the pea plastocyanin gene (PetE) promoter has been investigated in transgenic plants containing chimeric promoters fused to the -glucuronidase (GUS) reporter gene. This region enhanced GUS expression in leaves of transgenic tobacco plants when fused in either orientation to a minimal pea PetE promoter (-176 to +4) and in roots when fused in either orientation upstream or downstream of a minimal cauliflower mosaic virus 35S promoter (-90 to +5). The region was also able to enhance GUS expression in microtubers of transgenic potato plants when placed in either orientation upstream of a minimal class I patatin promoter (-332 to +14). Dissection of P268 revealed that cis elements responsible for enhancing GUS expression from the minimal PetE promoter were distributed throughout P268. Multiple copies of a 31 bp A/T-rich sequence from within P268 and of a 26 bp random A/T sequence were able to enhance GUS expression from the minimal PetE promoter, indicating that A/T-rich sequences are able to act as quantitative, non-tissue-specific enhancer elements in higher plants. Abbreviations: CaMV, cauliflower mosaic virus; GUS, -glucuronidase; HMG, high-mobility group; MAR, matrix-associated region; MU, methylumbelliferone; SAR, scaffold-associated region.     

A multiple-stimuli-responsive as-1-related element of parA gene confers responsiveness to cadmium but not to copper.

The expression of parA, an auxin-regulated gene expressed during the culture of tobacco (Nicotiana tabacum L.) mesophyll protoplasts, is induced by cadmium. To identify the cadmium-responsive element, we examined the parA promoter using the GUS reporter gene. Cadmium responsiveness was retained in a 5' deletion of the parA promoter to -78 bp, but it was nullified by further deletion to -49bp, which implies that the region -49 to -78 bp contained a cadmium-responsive element. This region contains a sequence similar to as-1, an enhancer sequence from the cauliflower mosaic virus 35S RNA promoter that binds the nuclear factor ASF-1. We named the sequence in the parA promoter pas. Gel-shift assays revealed that pas and as-1 compete for the same DNA-binding nuclear protein(s). Since pentamers of either pas and as-1 were able to confer cadmium responsiveness on a minimal promoter but mutant as-1 was not, we propose that pas and as-1 are involved in cadmium-responsive gene expression. Neither pas nor as-1 conferred responsiveness to copper. The specificity of this response, involving the function of as-1-related elements including pas, is discussed.     

Expression of insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers the cold tolerance to transgenic tobacco

To elucidate the function of antifreeze protein from Microdera puntipennis dzhungarica for freezing stress tolerance in plant, the construct of MpAFP149 gene with the signal peptide sequence responsible for secreting the native MpAFP149 into the apoplast space under control of a cauliflower mosaic virus 35S promoter was introduced into tobacco by Agrobacterium tumefaciens-mediated transformation. The observation of immunogold localization by TEM (transmission electron microscope) showed that the heterologous MpAFP149 protein was mainly distributed on the cell wall in apoplast of the transgenic tobacco plant. T1 generation transgenic tobacco plants displayed a more frost resistant phenotype and kept the lower ion leakage ratio and MDA (malondialdehyde) content in the leaves compared with wild-type ones at -1 degrees C for 3 days. The results showed that MpAFP149 provided protection and conferred cold tolerance to transgenic tobacco plant during freezing stress.     

转基因烟草表达西红花CSzCD基因产生西红花酸

为研究转基因烟草中产生西红花酸的可行性,在本研究中,西红花玉米黄素裂解酶（CSzCD）基因插入到pBI121载体的花椰菜花病毒（CaMV）35S启动子下游,通过农杆菌介导整合到烟草基因组中。通过Southern blotting 分析得到21株转基因烟草植株系; 转基因烟草叶片提取物Western blotting 和HPLC分析显示有西红花酸的产生,然而阴性对照中并没有发现西红花酸的存在。     

Construction of phosphomannose isomerase (PMI) transformation vectors and evaluation of the effectiveness of vectors in tobacco (Nicotiana tabacum L)

Phosphomannose isomerase (pmi) gene isolated from Escherichia coli allows transgenic plants carrying it to convert mannose-6- phosphate (from mannose), a carbon source that could not be naturally utilized by plants into fructose-6-phosphate which can be utilized by plants as a carbon source. This conversion ability provides energy source to allow the transformed cells to survive on the medium containing mannose. In this study, four transformation vectors carrying the pmi gene alone or in combination with the β-glucuronidase (gusA) gene were constructed and driven by either the maize ubiquitin (Ubi1) or the cauliflower mosaic virus (CaMV35S) promoter. Restriction digestion, PCR amplification and sequencing were carried out to ensure sequence integrity and orientation. Tobacco was used as a model system to study the effectiveness of the constructs and selection system. PMI11G and pMI3G, which carry gusA gene, were used to study the gene transient expression in tobacco. PMI3 construct, which only carries the pmi gene driven by CaMV35S promoter, was stably transformed into tobacco using biolistics after selection on 30 g 1(-1) mannose without sucrose. Transgenic plants were verified using PCR analysis. ABBREVIATIONS: PMI/pmi - Phosphomannose isomerase, Ubi1 - Maize ubiquitin promoter, CaMV35S - Cauliflower mosaic virus 35S promoter, gusA - β-glucuronidase GUS reporter gene.     

Comparing constitutive promoters using CAT activity in transgenic tobacco plants

The effectiveness of different promoters for use in transgenic tobacco was compared using a reporter gene expressing chloramphenicol acetyl transferase (CAT). Plasmids with CAT gene controlled by cauliflower mosaic virus 35S (CaMV 35S), rice actin1 (Ract1) and tobacco polyubiquitin (Tubi.u4) promoters were delivered into tobacco plants by Agrobacterium-mediated transformation. The Ract1 promoter, previously shown to be a strong promoter in rice and other monocots, failed to promote strong expression in tobacco. CAT expression was greatest from the vector carrying Tubi.u4 with a 5'UTR and leader intron without a ubiquitin monomer. In transgenic plants harboring the Tubi.u4 promoter, CAT expression was approximately twice that of the CaMV 35S promoter. Our results suggest that foreign genes under the control of a ubiquitin promoter devoid of monomer will be useful for high-level gene expression in tobacco.