The reporter gene was the uida gene of escherichia coli that encodes the enzyme. Transcription from the promoter was activated by crossing reporter plants with activator lines that expressed a chimeric transcription factor, lhg4. Expression and inheritance of gus gene in transgenic. Further developments may lead to routine in vivo analysis and fusion genetics. There are several ways to generate transgenic plants. Under normal conditions, the transgenic plants showed none of any. As expected, none of the gus plants showed any detectable gus activity data not shown.
Vascularspecific expression of gus and gfp reporter genes. Benefits of the gus gene reporter system in plants bitesize bio. Analysis of transgenic plants specifically expressing the egccrgfpgus construct. Fertile transgenic plants of datura innoxia were obtained through transformation of leaf tissue with disarmed agrobacterium tumefaciens strain lba 4404, harbouring a binary vector containing a chimeric fusion between the pea hsp 70 lp19 gene promoter and the gus reporter gene, and in vitro regeneration via organogenesis. The gus reporter gene system is already a powerful tool for the assessment of gene activity in transgenic plants. A transcription activation system for regulated gene. Since its description by jefferson, the gus gene fusion system has found extensive application in plant gene expression studies because of the enzyme stability and the high sensitivity and suitability of the.
Five out of those 14 lines were selected randomly to generate t1, followed by t2, and t2 respectively. Abstract the gus reporter gene system is already a powerful tool for the assessment of gene activity in transgenic plants. In vitro culture of petunia hybrida microspores and agrobacteriummediated transient expression of bglucuronidase gus reporter gene. The presented data indicate that the used 720bplong promoter region can provide replication dependent expression for the gus reporter gene in. Here, we tested the flpfrt recombination system in a construct containing a two gene cassette organization and examined its potential in transgenic arabidopsis and tobacco plants using a. B the expression of the gus gene was driven by the ibrbcs1 promoter in the leaves of transgenic plants. The selected transgenic arabidopsis plant xgd2v116 exhibited a correlation between uptake of pcb126 and pcb126induced gus activity. The process of developing transgenic plants without the presence of selectable marker genes or by use of more acceptable marker genes is regarded as clean gene technology. In each experiment 10 plants were transformed and on average 14 transgenic lines were recovered at the end of primary screening. Transgenic tomato, tobacco and arabidopsis plants containing the lat59 promoter region fused to gus also showed very high levels of gus activity in pollen.
Targetedgenomeeditingin nicotianatabacum induciblecrispr. The aim of this study was to observe the difference between the activities of introncontaining and intronless gus genes in transgenic tobacco plants and the. Some reporter genes such as the gfp, gus, and luc expression are fluorometric or colorimetric, where the expression of these genes could be observed. The p69b and p69c genes were shown to be induced following infection of tomato plants with p. Histochemical analysis of gus expression in leaf tissue. In situ detection of expression of the gus reporter gene. The sweet potato rbcs gene ibrbcs1 promoter confers high. Dna of the vector is preferentially accepted and the mcf can transform it into agrobacterium.
The apparent simplicity of the histochemical gus assay has been a major factor in the increase in articles. In molecular biology, a reporter gene often simply reporter is a gene that researchers attach to a regulatory sequence of another gene of interest in bacteria, cell culture, animals or plants. Transgenic plants with the gus reporter gene susan j. A widely applicable promoter system is described that allows a gene of interest to be activated in specific plant tissues after a cross between defined transgenic lines. Here we report that the promoters for chloroplast gs2 and cytosolic gs3a of pisum sativum confer nonoverlapping, cellspecific expression patterns on the. The pcambia2301 vector is recommended and can be provided prior to subcloning. Lets say that for one of the transgenic lines, i have only one positive plant pcr tested to contain the inserted gene. Gus gene reporter system is one of the popular and common plant gene. Cellspecific expression in transgenic plants reveals. Grapevine microshoots were transformed with the egccrgfpgus construct containing 1. Among the methods now available to localize the sites of gene expression in plant materials, reporter genes based on the gus uid a gene of escherichia coli, which encodes a.
Among the methods now available to localize the sites of gene expression in plant materials, reporter genes based on thegus uida gene ofescherichia coli, which encodes a. The gene rolb from the tdna of agrobacterium rhizogenes is capable of directing differentiation of roots in transfornned plant cells. In this model system, a heatshock inducible promoter was employed to control the expression of the flp recombinase gene. Due to the availability of gus and gfp reporter systems for plant transformants, cat is not commonly used. Among the methods now available to localize the sites of gene expression in plant materials, reporter genes based on thegus uida gene ofescherichia coli. The transgenic arabidopsis plants carrying a recombinant guinea pig g aryl hydrocarbon receptor ahrmediated. Gus is a good reporter for plants, as it does not occur naturally, and thus, has a low background. In order to use gus as a reporter of promoter activity, a transgenic plant containing the promotergusa chimeric gene is made. Ordering transgenic plants from the mature citrus facility mcf i. Recombinant aryl hydrocarbon receptors for bioassay of. Histological gus assays showed that transactivation occurred in all types of tissue and at all developmental stages.
No expression was detected from this promoter when placed upstream of a. The presence of the gus reporter gene in these transgenic plants was detected by pcr amplification, using gusspecific. Transgenic plants in order to use gus as a reporter of promoter activity, a transgenic plant containing the promotergusa chimeric gene is made. The gus gene reporter system is one of the popular and common plant reporter systems. Molecular techniques have been applied to an array of species, resulting in the generation of numerous transgenic plants. And this will result in the production of many markerfree transgenic plants that will be readily acceptable by the public. For experiments based on transgenic gus tgus, we used transformed plants of a.
This clearly demonstrated expression from these promoters in leaf epidermal cells. Report by international journal of agriculture and biology. Thus, other screening methods such as polymerase chain reaction pcr and reporter gene expression screening are used for better accuracy as an alternative screening method for transgenic plants. Recombinant human ahrmediated gus reporter gene assays for pcb congeners in transgenic tobacco plants in comparison with recombinant mouse and guinea pig ahrs. Uid a gene has become one of the most widely used reporter gene in plant molecular biology and microbiology. Local and systemic induction of reporter gene expression driven byp69b and p69c promoters. Such genes are called reporters because the characteristics they confer on organisms expressing them are easily identified and measured, or because they are selectable markers.
The term gus staining refers to the most common of these, a histochemical technique. Chloroplast and cytosolic isoforms of glutamine synthetase gs. Analysis of transgenic plants containing these chimeric genes provided definitive proof that the chia coding region is regulated by two distinct promoters designated pai and paz. Expression of two tissuespecific promoters in transgenic. Impact of ubiquitous inhibitors on the gus gene reporter. In vitro culture of petunia hybrida microspores and.
The cat gene producing chloramphenicol acetyl transferase cat is a widely used reporter gene in mammalian cells. For commercialization of transgenics, the gus reporter gene, not gfp, should be used with nptii selection. Gus uida reporter gene expression for two sugarcane polyubiquitin promoters, ubi4 and ubi9, was compared to expression from the maize ubi1 promoter in stable transgenic rice only ubi9 and sugarcane ubi4 and ubi9. Further developments may lead to routine in vivo analysis and fusion. Gene reporters enable valuable insight into gene expression. To understand how this induction is regulated and where does it take place in the infected plant, we constructed different chimeric promoter fusions with the bacterial. However, some workers continue to use cat by a sensitive radioactive assay, for the detection of the reporter gene cat. Transgenic plants and cells containing the chsgus or other defense gene promoterreporter gene fusions may therefore provide convenient. Progress in plant genetic engineering has been spectacular since the recovery of the first transformed plants in the early 1980s. Several kinds of gus reporter gene assay are available, depending on the substrate used. Effects of light on the expression of the ibrbcs1 gene or gus gene. Local and systemic induction of two defenserelated. Ubi9 drove highlevel gus expression, comparable to the maize ubi1.
Plant defense gene promoterreporter gene fusions in transgenic. Expression of gus activity in the reporter plants was very low, but high gus activity could be induced by introduction of gene vi, either by crossing with plants containing gene vi as a transgene or by infection with camv. We therefore examined these cells for the presence of chloroplasts and reexamined expression from the rbcs promoter in epidermal cells. Analysis of the progeny by gus staining and pcr amplification revealed that all of the 5fcresistant r1 plants were coda free, and that the coda gene segregated independently of the gus gene. Meristem, cell division and s phasedependent activity of.
Transgenic cotton plants t 0 were regenerated from two independent cell lines transformed with the ghsp gus gene construct. A the expression levels of the ibrbcs1 gene in the leaves of sweet potato grown under a 14h10h lightdark photoperiod. In order to use gus as a reporter of promot er activity, a transgenic plant containing the promoter gus a chimeric gene is made. The gus reporter system is a reporter gene system, particularly useful in plant molecular biology and microbiology. The gus reporter system in flower development studies. Gus plants grown in the presence of mc exhibited a clear induction of gus activity to varying degrees. The reporter gene expression was low during mitosis when accumulation of the transcripts of a btype mitotic cyclin cycb1.
We also showed that both promoters can function independently and that the chia pai promoter is expressed in limb. Since then, the uida gene commonly referred to as the gus gene has become one of the most widely used reporter genes in plant molecular biology. Gus reporteraided analysis of the promoter activities of arabidopsis. The reporter gene was the uid a gene of eascherichia coli that encode the enzyme. However, analysis of gus assays is often complex in plant cells, as gus gene expression is affected by various biochemical, molecular and biological factors that render its use as a reporter gene susceptible to limitations. Gus, is short for glucuronidase, an enzyme in the bacterium e. The ratelimiting bacterial iaabiosynthetic gene iaam and the reporter gene for.
We also demonstrate the use of a linked reporter gene to visualize the expression of the iaa genes. Read the independent project description in the lab manual. Gene fusions between the promoter of rolb and the gus reporter gene encoding e. Treatment of these transgenic plants with hormones and stresses showed that atacs genes were differentially regulated, suggesting that each atacs gene had a. Karcher department of biological sciences purdue university west lafayette, in 4790792 phone. Pollenspecific gene expression in transgenic plants. In situ detection of expression of the gus reporter gene in transgenic. For many plant species, the easiest method is agrobacteriummediated gene transfer. Histological analysis of the expression of agrobacterium. With 4mug as a substrate, plants containing the guswt gene showed. Effects of biosurfactants on assays of pcb congeners in. Impact of ubiquitous inhibitors on the gus gene reporter system. Journal of environmental science and health, part b. The promoter, pop, consists of lac operators cloned upstream of a minimal promoter.
Benefits of the gus gene reporter system in plants. Gus, have been the most widely used during the last ten years. Business, international agrobacterium tumefaciens genetic aspects health aspects physiological aspects botanical research gene expression research hostparasite. Assay the gus reporter gene in the transgenic blue plants.456 1215 232 113 339 1497 948 868 1101 1495 1370 416 66 800 752 1321 1140 480 1514 843 1207 66 656 542 580 423 402 552 442 922 1464 1435 1231 126 897 1029 1032 1284 377 1276 1097 441 1168 1296