Navigation Links
Using the VersaFluor Fluorometer to Quantitate GUS Expression in Plant Tissues Bombarded With the Biolistic PDS-1000/He System

Nicholas N. Lyssenko and Mark L. Tucker, Soybean and Alfalfa Research Lab, USDA, ARS, Bldg. 006, BARC-West, Beltsville, MD, USA 20705


Introduction
In 1987 Jefferson et al. described the use of β-glucuronidase (GUS) as a marker to study gene expression in plants. Expression of GUS activity in plant tissue can be quantified by fluorometry or assayed qualitatively by histochemistry. The GUS enzyme cleaves the commercially available substrate 4-methylumbelliferyl-β-D-glucuronic acid (MUG) to produce fluorescent methylumbelliferone (MU). The VersaFluor fluorometer, when equipped with the simple-to-install 360 excitation and 460 emission filters, has the necessary sensitivity to accurately measure MU in plant tissues. Quantification of GUS activity using the VersaFluor fluorometer is relatively inexpensive and reliable. We have used the instrument to quantify transient expression of a CaMV 35S-GUS construct in bean abscission zones. Abscission is the process by which plants shed organs. The bean abscission cellulase (BAC) accumulates tissue specifically in induced abscission zones. GUS expression is used as an internal control in experiments designed to identify regulatory regions in the BAC gene promoter. An example of the approach used and results obtained is described below. In addition to quantification of GUS activity, we use the VersaFluor fluorometer equipped with the same set of filters to quantify the DNA preparations. The fluorescent dye for DNA quantification is Hoechst 33258 from Bio-Rad.


Materials and Methods
The promoter to be tested for hormonal or tissue-specific expression is fused to the luciferase gene taken from the pDO432 plasmid (Ow et al., 1986; see Koehler et al., 1996, for details of promoter construction). The GUS gene is fused to an enhanced 35S promoter (Kay et al., 1987). An accurate measure of the concentration of the gene constructs is determined with the VersaFluor fluorometer using the Hoechst 33258 dye from Bio-Rad. The two constructs are then co-precipitated at constant and equal-molar concentrations onto gold particles (Figure 1A). Plant material is bombarded with the gold particles using the Biolistic PDS-1000/He particle gun (Bio-Rad) as depicted in Figure 1B.

Following particle bombardment and a 48-hour incubation period in ethylene (a plant hormone that induces abscission), approximately 1 mm of tissue is harvested from the upper surface of the bombarded explants (abscission zones, petioles and stems), and frozen separately in liquid nitrogen (Figure 1B). The protein extraction procedure is modified from the original protocol described by Jefferson et al. (1987) to allow the assay of cellulase, luciferase, and GUS activity from the same tissue extract. Approximately 150 mg of plant tissue is ground in liquid nitrogen, thawed in 450 l of extraction buffer (0.1 M sodium phosphate, pH 7.8, 1% Triton X-100, 2 mM EDTA, 1 mM DTT and 0.1% BSA), vortexed and then centrifuged at 10,000 rpm in a microcentrifuge for 4 minutes.

The supernatant is then transferred to new tubes in 75 l aliquots for measurement of cellulase and luciferase activity as described elsewhere (Koehler et al., 1996). For GUS assay, 75 l of the supernatant is combined with 25 l of a GUS adjustment buffer (0.4 M citrate buffer pH 5.5, 34 mM EDTA and 17 mM DTT) and the combined mixtures incubated at 55 C for 30 minutes. The GUS marker enzyme, originally from E. coli, is resistant to heat inactivation at 55 C; however, heating significantly reduces endogenous GUS-like activity in plant extracts. Following incubation, the mixture is combined with 20 l of methanol, which further reduces background GUS-like activity (Kosugi et al., 1990), and 12.5 l of 10 mM MUG. Immediately after addition of MUG, 10 l of the mixture is removed and combined with 2 ml of 0.2 M sodium carbonate to stop GUS conversion of MUG to MU. These tubes are labeled T0. The remaining mixture is incubated at 37 C overnight and the T0 tubes stored in the dark at room temperature. Following incubation at 37 C, 10 l of the mixture is combined with 2 ml of 0.2 M sodium carbonate and the tube labeled T12.

MU standards are prepared at 50, 100 and 500 nM in 0.2 M sodium carbonate. The VersaFluor fluorometer is warmed for 15 minutes before conducting the assay. The instrument has low, medium and high gains. The range of MU standards described above is best measured at a medium gain.

The instrument is zeroed with a blank consisting of 0.2 M sodium carbonate. First, the highest standard, 500 mM, is measured and set to equal 5,000 relative units with the range option of the fluorometer. Next, the two remaining standards are measured to ascertain a linear response. Finally, the fluorescence of extracts in the T0 and T12 tubes is measured and recorded.


Results and Discussion
Fusion of the enhanced CaMV 35S promoter (Kay et al., 1987) to the GUS reporter gene creates a gene construct (D35S) that expresses GUS constitutively in transformed plant tissues. Inclusion of this construct is used as an internal control to reduce variability in all our particle bombardment experiments (Figure 1A). The expression results (Figure 2B) are reported as the ratio of luciferase to GUS activity normalized to 100% for BKm3 expression in abscission zones. Expression of luciferase from the test promoter, the BKm3 construct (Figure 1A), relative to GUS expression from D35S (i.e., luciferase to GUS ratio) in stems and petioles is approximately 10% of the expression in abscission zones (Figure 2B). This pattern of expression is very similar to that for native cellulase activity measured in the same extracts collected from particle bombarded stems, petioles and leaf abscission zones (LAZ) (Figure 2A).

The VersaFluor fluorometer is inexpensive and easy to use. It has the necessary sensitivity to reproducibly measure transient GUS activity in particle gun bombarded plant tissues. In addition, using the Hoechst 33258 dye to quantitate DNA by the VersaFluor fluorometer is reliable, simple, and accurate.


References
1. Jefferson, R. A., Kavanagh, T. A. and Bevan, M. W., EMBO 6, 3901-3907 (1987).

2. Kay, R., Chan, A., Daly, M. and McPherson, J., Science, 236, 1299-1302 (1987).

3. Koehler, S. M., Matters, G. L., Nath, P., Kemmerer, E. C. and Tucker, M. L., Plant Molec. Biol., 31, 595-606 (1996).

4. Ow, D. W., Wood, K. V., DeLuca, M., DeWet, J. R., Helinski, D. R., and Howell, S. H., Science, 234, 856-859 (1986).


Triton is a trademark of Rohm and Haas. Biolistic is a trademark of E.I. Du Pont de Nemours and Co. Biolistic technology is exclusively licensed to Bio-Rad Laboratories.


back to top
'"/>

Source:


Page: All 1 2 3 4

Related biology technology :

1. Low Abundance cDNA Cloned Using Stratagenes Human Universal cDNA Library
2. Oil-Free PCR Using the Hot Top Assembly
3. Functional Cloning Using ViraPort Retroviral cDNA Expression Libraries
4. Signal Transduction Reporting Systems Using Cis-Acting Enhancer Elements
5. Using XL1-Red Mutator Strain to Generate Esterase Variants
6. Detect Released Oligosaccharides Using the Eagle Eye II Still Video Imaging System
7. Comparison of Growth Techniques and Media for the Purpose of Plasmid Isolation from E. coli Using the Eppendorf Perfectprep Plasmid Mini Kit
8. Genomic DNA Extraction from Buccal Swabs Using the Perfect gDNA Blood Mini Kit
9. Genomic DNA Extraction from Buffy Coat Using the Perfect gDNA Blood Mini Kit
10. Isolation of Genomic DNA from Saliva Using the Perfect gDNA Blood Mini Kit
11. Protocol for RNA Isolation Using TRIzol Reagent with Phase Lock Gel-Heavy
Post Your Comments:
(Date:9/18/2014)... OMICS Group International invites researchers, authors and ... ‘Open Access Week’ by actively contributing their research ... Journals from 20-26th October, 2014. , Pharmaceutical ... accelerating with a tremendous speed to provide improved ... industry is gearing with innovation and technological revolution ...
(Date:9/18/2014)... 2014 SeqLL Inc., the owner ... announced today the closing of a $1M Series-A ... Genomic Diagnostic Technologies, will increase the power of ... Founded in March 2013 by Daniel Jones, a ... Helicos BioSciences, SeqLL has continued to offer Helicos’ ...
(Date:9/18/2014)... group of rare diseases that afflict skeletal growth ... hits at the fetal stage and is caused ... encoding fibroblast growth factor receptor 3 (FGFR3) has ... thanatophoric dysplasia (TD), a skeletal dysplasia that cause ... lethal, and achondroplasia (ACH), which causes stunted growth ...
(Date:9/18/2014)... Furnishing a research lab can be pretty expensive. Now ... University has published an open-source library of designs that ... used piece of equipment: the syringe pump. , Syringe ... as for drug delivery or mixing chemicals in a ... of dollars. , Joshua Pearce and his team of ...
Breaking Biology Technology:Pharma and Omics Journals to Address Global Challenges during Open Access Week 2Pharma and Omics Journals to Address Global Challenges during Open Access Week 3SeqLL Closes Series-A Funding Round for Expansion of Single Molecule Sequencing 2Researchers use iPS cells to show statin effects on diseased bone 2Researchers use iPS cells to show statin effects on diseased bone 3Doing science just got cheaper -- and faster 2
... Calif., June 6 InterMune, Inc.,(Nasdaq: ITMN ) ... Financial Officer of InterMune, will present at the Goldman ... on,June 10, 2008 at 8:05 a.m. PDT (11:05 a.m. ... Needham & Company, LLC Biotechnology and,Medical Technology Conference in ...
... 5 BioTrends Research Group, Inc.,released its third ... management of psoriasis. This report is based on,responses ... Dermatologists in April,2008., According to the study, ... use of biologics for the treatment of psoriasis ...
... BioTrends Research Group, Inc. is,pleased to announce the ... is based on the results of an on-line ... France, Germany, Italy, and Spain.,The report focuses on ... In the ESA market, Amgen,s Aranesp is ...
Cached Biology Technology:InterMune to Present at Goldman Sachs Healthcare and Needham & Co. Biotechnology and Medical Technology Conferences 2Dermatologists Report Increased Use of Biologics in the Treatment of Psoriasis 2New Study by BioTrends Highlights Practice Management Shifts Among European Nephrologists 2
(Date:9/18/2014)... that when people are too stressed they are often ... Brain Mind Institute (BMI) at EPFL have just highlighted ... chronic stress and the loss of social skills and ... a synaptic regulatory molecule in the brain. This was ... . , Carmen Sandi,s team went to look ...
(Date:9/18/2014)... the roaches of the fish world, are the ideal ... body shape. They,ve moved from the ocean into tens ... world, each time changing their skeleton to adapt to ... freshwater populations of sticklebacks now have turned up one ... that a simple change in that gene,s regulation in ...
(Date:9/18/2014)... the ultimate form of camouflage: you don,t just blend ... is not as uncommon as you might think. Kathryn ... explains that the larval life stages of many marine ... the anatomy that most creatures cannot make transparent. Feller ... shield each individual eye unit with an opaque pigment ...
Breaking Biology News(10 mins):How stress tears us apart 2Counting fish teeth reveals regulatory DNA changes behind rapid evolution, adaptation 2Counting fish teeth reveals regulatory DNA changes behind rapid evolution, adaptation 3Transparent larvae hide opaque eyes behind reflections 2
... the results of the Oats at 10 Years study, marking ... claim that oats, as part of an overall heart healthy ... written by Mark B. Andon, PhD, and James W. Anderson, ... claim, theorizing that foods containing whole-oat sources of soluble fiber ...
... University bioengineer Jennifer West is being recognized today with ... the O,Donnell Award from The Academy of Medicine, Engineering ... given for excellence in medical, scientific and engineering research, ... statue. West, a pioneer in the field of biomaterials ...
... of Alberta for those who may have overindulged during the recent ... a way to reduce the amount of bad cholesterol and fatty ... metabolizes, a key discovery that could lead to new drugs to ... disease related to obesity. In a series of recently ...
Cached Biology News:Rice's West honored as one of Texas' best researchers 2Rice's West honored as one of Texas' best researchers 3University of Alberta researchers report breakthrough in lowering bad cholesterol, fatty acid levels 2
... The Sub-Plates are E.Coli glycerol stocks contained ... contains 50 cDNA clones with approximately 5,000 ... the a master plate well, the corresponding ... PCR to identify the positive well(s). Cells ...
... 0.9 ml per liter of urine will give ... or BMD dipstick. ,Certification: The Donors are ... found to be NEGATIVE for HbsAg, HCV, HIV-1 ... approved FDA methods. THIS MATERIAL IS SOLD FOR ...
One controller two vessel system suitable for early stage of fermentation, and small scale, Application : cultivation of microorganisms and human or animal cells...
... light production by luciferase-cDNA transfected cell lines ... in the development of reporter gene assays ... an indicator for the transcription of other ... is due to advantages such as high ...
Biology Products: