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1.
Transferred DNA (T-DNA) tagging is a powerful tool for tagging and in planta characterization of plant genes on a genome-wide scale. An improved promoter tagging vector is described here, which contains the codon-optimized luciferase (luc+) reporter gene 31 bp from the right border of the T-DNA. Compared to the wild-type luciferase gene, this construct provides significantly increased reporter gene expression and a 40 times higher tagging frequency. The utility of the construct is demonstrated in banana, a tropical monocot species, by screening embryogenic cell colonies and regenerated plants with an ultrasensitive charged-coupled device (CCD) camera. The improved vector resulted in a luciferase activation frequency of 2.5% in 19,000 cell colonies screened. Detailed molecular analysis of flanking DNA sequences in a tagged line revealed insertion of the luciferase tag in a novel gene with near-constitutive expression. 相似文献
2.
Recently the gfp (green fluorescent protein) gene from the jellyfish Aequoria victoria has been widely used as a reporter gene. In this study mini-transposons, named as mini-Tn5gfp, were constructed by subcloning
the gfp gene into a transposon Tn5. To improve the expression level of the gfp gene, tandom array of gfp gene was obtained. The constructs were successfully used in tagging target microorganisms by transposition. The level of
GFP expression was found to be closely correlated with the copy number of the gfp transposed. These constructs will facillitate
not only efficient tagging of whole organism but also genetic marking of target genes by transposition. 相似文献
4.
Transposable elements have been used as an effective mutagen and as a tool to clone tagged genes. Insertion of a transposable element into a gene can lead to loss- or gain-of-function, changes in expression pattern, or can have no effect on gene function at all, depending on whether the insertion took place in coding or non-coding regions of the gene. Cloning transposable elements from different plant species has made them available as a tool for the isolation of tagged genes using homologous or heterologous tagging strategies. Based on these transposons, new elements have been engineered bearing reporter genes that can be used for expression analysis of the tagged gene, or resistance genes that can be used to select for knockout insertions. While many genes have been cloned using transposon tagging following traditional forward genetics strategies, gene cloning has ceased to be the rate-limiting step in the process of determining sequence–function relations in several important plant model species. Large-scale insertion mutagenesis and identification of insertion sites following a reverse genetics strategy appears to be the best method for unravelling the biological role of the thousands of genes with unknown functions identified by genome or expressed sequence tag (EST) sequencing projects. Here we review the progress in forward tagging technologies and discuss reverse genetics strategies and their applications in different model species. 相似文献
6.
Summary This article reviews techniques for gene identification and cloning in allohexaploid bread wheat ( Triticum aestivum L.). Gene identification and cloning in wheat are complicated by the large size and high redundancy of the genome. Both classical
mutagenesis and transposon tagging are important tools for the study of grain dormancy and plant hormone signaling in wheat.
While classical mutagenesis can be used to identify wheat mutants with altered hormone sensitivity, it can be difficult to
clone the corresponding genes. We review the techniques available for gene identification in wheat, and propose that transposon-based
activation tagging will be an important tool for wheat genetics. 相似文献
7.
As part of a gene tagging strategy to study the developmental regulation of patterns of plant gene expression, a promoterless uidA ( gus A) gene, encoding the -glucuronidase (GUS) reporter, was introduced into populations of tobacco, Arbidopsis and potato by Agrobacterium-mediated gene transfer. The objective was to generate random functional fusions following integration of the gusA gene downstream of native gene promoters. We describe here a detailed analysis of levels and patterns of gusA activation in diverse organs and cell types in those populations. gusA activation occurred at high frequency in all three species, and unique patterns of fusion gene expression were found in each transgenic line. The frequency of gusA activation was differentially blased in different organs in the three species. Fusion gene activity was identified in a wide range of cell types in all organs studied, and expression patterns were stably transmissible to the T 2 and T 3 progeny. Developmentally-regulated and environmentally-inducible expression of gusA is described for one transgenic line. Phenotypic variants were detected in the transgenic population. These results demonstrate the potential of T-DNA insertion as a means of creating functional tags of genes expressed in a wide spectrum of cell types, and the value of the approach as a complement to standard T-DNA insertional mutagenesis and transposon tagging for developmental studies is discussed. 相似文献
8.
Genetic transformation of plants offers the possibility of functional characterization of individual genes and the improvement of plant traits. Development of novel transformation vectors is essential to improve plant genetic transformation technologies for various applications. Here, we present the development of a Gateway-compatible two-component expression vector system for Agrobacterium-mediated plant transformation. The expression system contains two independent plasmid vector sets, the activator vector and the reporter vector, based on the concept of the GAL4/UAS trans-activation system. The activator vector expresses a modified GAL4 protein (GAL4-VP16) under the control of specific promoter. The GAL4-VP16 protein targets the UAS in the reporter vector and subsequently activates reporter gene expression. Both the activator and reporter vectors contain the Gateway recombination cassette, which can be rapidly and efficiently replaced by any specific promoter and reporter gene of interest, to facilitate gene cloning procedures. The efficiency of the activator–reporter expression system has been assessed using agroinfiltration mediated transient expression assay in Nicotiana benthamiana and stable transgenic expression in Arabidopsis thaliana. The reporter genes were highly expressed with precise tissue-specific and subcellular localization. This Gateway-compatible two-component expression vector system will be a useful tool for advancing plant gene engineering. 相似文献
9.
We describe a new Renilla reniformis luciferase reporter gene, RiLUC, which was designed to allow detection of luciferase activity in studies involving Agrobacterium-based transient expression studies. The RLUC gene was altered to contain a modified intron from the castor bean catalase gene while maintaining consensus eukaryotic splicing
sites recognized by the plant spliceosome. RLUC and RiLUC reporter genes were fused to the synthetic plant SUPER promoter. Luciferase activity within agrobacteria containing the SUPER- RLUC construct increased during growth in culture. In contrast, agrobacteria harboring the SUPER- RiLUC gene fusion showed no detectable luciferase activity. Agrobacteria containing these gene fusions were cotransformed with
a compatible normalization plasmid containing a cauliflower mosaic virus 35S promoter (CaMV) joined to the firefly luciferase
coding region ( FiLUC) and infused into tobacco leaf tissues through stomatal openings. The kinetics of luciferase production from the RLUC or RiLUC reporters were consistent, with expression of the RiLUC gene being limited to transiently transformed plant cells. RiLUC activity from the reporter gene fusions was measured transiently and within stably transformed tobacco leaf tissues. Analysis
of stably transformed tobacco plants harboring either reporter gene fusion showed that the intron altered neither the levels
of luciferase activity nor tissue-specific expression patterns driven by the SUPER promoter. These results demonstrate that
the RiLUC reporter gene can be used to monitor luciferase expression in transient and stable transformation experiments without interference
from contaminating agrobacteria. 相似文献
11.
Potato is an autotetraploid crop plant that is not very amenable to the deployment of transposon tagging for gene cloning
and gene identification. After diploidisation it is possible to get potato genotypes that grow well, but they are self-incompatible.
This prevents the production of selfed progeny that are normally used in gene tagging approaches to select for parental lines
with the target gene to be tagged in a homozygous stage. We describe here an alternative selection method for directed transposon
tagging for a gene of interest in a heterozygous background. Diploid potato plants with a Ds transposon linked to the desired gene of interest (the Phytophthora
infestans R1 resistance locus) in a heterozygous stage were used for the development of this directed transposon tagging strategy. After
crossing to a diploid Ac transposon-containing genotype, 22 ’interesting’ seedlings (R1Ds/r–; Ac/–) were selected that showed active Ds transposition as displayed by DNA blot hybridisation, empty donor site PCR and sequencing. Protoplast isolation and the use
of the hygromycin gene as a cell-specific selection marker of Ds excision enabled the direct selection of Ds excision sectors in these highly chimaeric seedlings. This somatic selection of Ds transpositions and the regeneration through protoplasts resulted in the development of a large population of almost 2000
hygromycin-resistant plants. Southern blot analysis confirmed the insertion of Ds at independent positions in the genome. Every selected plant displayed independent Ds excisions and re-insertions due to the expression of the Ac transposase throughout development. This population, which is developed from seedlings with the desired R1 gene in a heterozygous stage, is directly useful for searching for transposon-tagged R1 mutants. In general, this approach for selecting for somatic transpositions is particularly suitable for the molecular isolation
of genes in a heterozygous crop like potato.
Received: 29 November 1999 / Accepted: 30 December 1999 相似文献
12.
We describe several new modifications of the Aequorea victoria green fluorescent protein (GFP) gene. The erGFP5
INTreporter gene combines the PIV2 intron from gus
INTand LUC
INTwith the ER-localized mGFP4-ER gene. The erGFP6
INT, erGFP7 INT, and erBFP8
INTgenes also include the fluorophore and solubility modifications of smGFP, smRS-GFP, and smBFP, respectively. A parallel set
of reporter genes ( erGFP5, erGFP6, erGFP7, and erBFP8) is otherwise identical to the respective erGFP
INTgenes but lacks the PIV2 intron. The intron-containing genes are expressed in plant cells but not in bacteria, allowing detection
of plant cell expression in the presence of Agrobacterium during the early stages of transformation. Transient expression of the erGFP
INTand erGFP genes is comparable in tobacco and maize suspension culture protoplasts, indicating that the PIV2 intron is spliced effectively
in both monocotyledonous and dicotyledonous plant species. 相似文献
14.
In a transposon tagging experiment, lines of Antirrhinum majus exhibiting both race-specific resistance (homozygous for the dominant R gene) to the rust pathogen Puccinia antirrhini and a high frequency of transposition were crossed with homozygous susceptible lines. From a screen of 11153 F 1 progeny, 15 individuals were detected showing susceptibility to rust race α. Six of these exhibited a susceptibility phenotype (classified as type B) not previously observed. A control experiment involving the same tagging strategy but employing lines that do not exhibit high rates of transposition did not yield any susceptible mutants from a screen of 6243 progeny. In experiments on the heritability and stability of the mutation, the six plants exhibiting susceptibility phenotype B produced progeny in which the R locus had reverted to an active form (i.e. some of the progeny were resistant), a classic characteristic of transposon-tagged plant genes. Reversion was shown to occur somatically, and its rate was temperature dependent. Inheritance studies showed that the mutations in two of the susceptible plants from the tagging protocol map at, or very close to, the race α-specific resistance gene. The results are consistent with the transposon tagging of a race-specific gene for rust resistance. 相似文献
16.
A novel Agrobacterium tumefaciens-mediated transient expression assay (AmTEA) was developed for young plants of different cereal species and the model dicot
Arabidopsis thaliana. AmTEA was evaluated using five promoters (six constructs) and two reporter genes, gus and egfp. The constitutive 35S promoter and the promoter of the rice glutaredoxin gene showed gus and egfp expression in the cereals analyzed in the present study. A promoter for the DEAD-box RNA helicase family protein gene from
Arabidopsis showed similar expression patterns of reporter genes in stable transgenic lines as well as in transient expression lines
of Arabidopsis. Agrobacterium tumefaciens co-cultivation and plant incubation times were optimized using 35S and the rice expressed protein gene promoter (R2-273).
The possibility of non-specific expression of the reporter genes was ruled out by using the antibiotic carbenicillin and the
comparison of expression of the reporter genes driven by full-length and truncated R2-273 promoters. AmTEA considerably reduced
time, space, labor, and cost requirements. Ease of use with stress treatments is another major advantage of this method. AmTEA
can be automated and used for large-scale studies to decipher promoter and gene functions with the ultimate goal to enhance
the performance of cereal crops against biotic and abiotic stresses. 相似文献
17.
A promoter tagging program in the legume Lotus japonicus was initiated to identify plant genes involved in the nitrogen-fixing symbiosis between legumes and rhizobia. Seven transformed plant lines expressing the promoterless reporter gene uidA (beta-glucuronidase; GUS) specifically in roots and/or nodules were identified. Four of these expressed GUS in the roots only after inoculation with nodule-forming Mesorhizobium loti. In one line (T90), GUS activity was found in the root epidermis, including root hairs. During seedling growth, GUS expression gradually became focused in developing nodules and disappeared from root tissue. No GUS activity was detected when a non-nodulating mutant of M. loti was used to inoculate the plants. The T-DNA insertion in this plant line was located 1.3 kb upstream of a putative coding sequence with strong homology to calcium-binding proteins. Four motifs were identified, which were very similar to the "EF hands" in calmodulin-related proteins, each binding one Ca2+. We have named the gene LjCbp1 (calcium-binding protein). Northern (RNA) analyses showed that this gene is expressed specifically in roots of L. japonicus. Expression was reduced in roots inoculated with non-nodulating M. loti mutants and in progeny homozygous for the T-DNA insertion, suggesting a link between the T-DNA insertion and this gene. 相似文献
18.
The proper use of a marker gene in a transformation process is critical for the production of transgenic plants. However,
consumer concerns and regulatory requirements raise an objection to the presence of exogenous DNA in transgenic plants, especially
antibiotic-resistant genes and promoters derived from viruses. One approach to overcome this problem is the elimination of
marker genes from the plant genome by using several site-specific recombination systems. We propose an alternative method
to solve this problem using a marker gene exclusively derived from the host plant DNA. We cloned a genomic DNA fragment containing
regulatory and coding sequences of acetolactate synthase (ALS) gene from rice, and mutagenized the ALS gene into a herbicide-resistant
form. After transfer of this construct to the rice genome, transgenic plants were efficiently selected with a herbicide, bispyribac-sodium
salt, which inhibits the activity of wild type ALS. We also analyzed the regulatory feature of the rice ALS gene promoter
with the gusA reporter gene and revealed that GUS expression was observed constitutively in aerial parts of rice seedlings and root tips.
The marker system consisted exclusively of host plant DNA and enabled efficient selection in a monocot crop plant, rice. The
selection system can potentially be applied to generate transgenic plants of other crop species and can be expected to be
publicly acceptable. 相似文献
19.
The use of reporter genes to characterise sequence elements that act to regulate gene expression in transgenic plants has been vital to the development of foreign gene expression strategies for use in cereal transformation. The gusA locus of Escherichia coli, which encodes the enzyme -glucuronidase (GUS), is by far the most popular reporter gene used in plant transformation. In this paper we extend the utility of the GUS reporter gene system in cereal transformation by describing and evaluating a number of novel constructs suitable for use in direct gene transfer experiments. These plasmids are all available from the Molecular Genetic Resource Service of the Center for the Application of Molecular Biology to International Agriculture. 相似文献
20.
在植物功能基因组学的研究中,插入突变已成为迅速识别和研究标签基因的一个有效遗传工具.本文介绍了T-DNA标签的概念及应用前提,详细论述了T-DNA标签在大规模植物基因功能分析中的应用以及使用启动子和增强子诱捕技术分离时空特异性启动子和表达基因,另外还分析了利用其特殊形式激活标签进行基因克隆和功能分析的优越性,并展望了T-DNA标签的应用前景. 相似文献
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