Transcript Brennan, Alex, Matt SRS Sem 1

The Stress Effects on RNAi
Expression when Stressing
Wild-type and Mutant
Strains of Arabidopsis With
Calcium Nitrate
By: Brennan Ramos, Matthew Newell
Alex Jackson
Monarch High School
Question
What are the effects on RNAi expression
when stressing wild type and mutant strains
(RDR126, DCL234) of Arabidopsis with
calcium nitrate?
Why use Arabidopsis?
•
•
•
•
•
•
Short life cycle 6-8 weeks
Small convenient to grow plant
Easily matched environmental conditions
Small genome
Has been completely genetically mapped
Closely resembles genes in crop plants
RDR6 and DCL4 Mutants
• Arabidopsis contains the genes for RDR 1-6,
and DCL genes 1-4.
• RDR - RNA dependant polymerase.
• DCL - Dicer Like
RNAi is the immune response of the
plant. The RISC complex cleaves
viral looking double stranded RNA
within the cytoplasm of the cell so
that the enzyme/protein can never be
produced.
Hypothesis
•
•
•
If there is a relationship between RNAi expression and
environmental conditions, and we stress wild type and
two other mutant Arabidopsis strains with calcium
nitrate, we should see a difference in phenotypic
expression.
We predict that under the same stress conditions we
will see differences in the development of the wild type
and mutant strains.
These differences may be in the form of changes in
length of developmental stages, number of leaves, size
and shape of leaves, and color of leaves.
Methods
• Used a magenta box system for wild type and two triple
knockout mutant plants to stress both using calcium nitrate.
• Our controls will be all three types of Arabidopsis plants
grown in agar, and our test groups will be all three types of
plants stressed with calcium nitrate.
• All plants will receive 16 hours of light and 8 hours of dark.
• We will stress with 1 and .25 molar calcium nitrate.
• We will follow the MSDS Safety Guidelines for calcium
nitrate. All plants will be autoclaved and disposed of through
the University of Colorado Denver.
Data
Plant Type
Tallest Height (mm)
# of Leaves
Color
Bolting?
DCL-234
1
2
Green
No
1
RDR-126
2
2
Green
No
1/28/2010
Wild Type
0
0
None
No
DCL-234
5
2
Green
No
2
RDR-126
6
2
Green
No
2/1/2010
Wild Type
1
2
None
No
DCL-234
6
2
Green
No
3
RDR-126
8
2
Green
No
2/6/2010
Wild Type
Mold
Mold
Mold
Mold
DCL-234
8
2
Purple
No
4
RDR-126
10
2
Green
No
2/15/2010
Wild Type
Mold
Mold
Mold
Mold
DCL-234
12
4
Purple
No
5
RDR-126
16
4
Purple
No
2/25/2010
Wild Type
Mold
Mold
Mold
Mold
Data
DCL-234
12
4
Purple
Yes
6
RDR-126
28
4
Purple
No
3/1/2010
Wild Type
Mold
Mold
Mold
Mold
DCL-234
19
4
Purple
Yes
7
RDR-126
40
4
Purple
Yes
3/5/2010
Wild Type
Mold
Mold
Mold
Mold
DCL-234
23
4
Purple
Yes
8
RDR-126
42
4
Purple
Yes
3/8/2010
Wild Type
Mold
Mold
Mold
Mold
DCL-234
35
4
Purple
Yes
9
RDR-126
43
4
Purple
Yes
3/11/2010
Wild Type
Mold
Mold
Mold
Mold
DCL-234
46
4
Purple
Yes
10
RDR-126
47
4
Purple
Yes
3/15/2010
Wild Type
Mold
Mold
Mold
Mold
Data
DCL-234
51
4
Purple
Yes
11
RDR-126
54
4
Purple
Yes
3/19/2010
Wild Type
Mold
Mold
Mold
Mold
DCL-234
51
4
Purple
Yes
12
RDR-126
55
4
Purple
Yes
4/1/2010
Wild Type
Mold
Mold
Mold
Mold
Height of Plants
Plant Height (mm)
60
Height (mm)
50
40
DCL-234
30
RDR-126
Wild Type
20
10
0
1
2
3
4
5
6
7
Measurments
8
9
10
11
12
Pictures
Pictures
Pictures
Conclusion
• It seems that there was a lot of mold growth in only the Wild Type
plants which leads us to believe that mutations in RDR and DCL give
those plants more resistance against mold..
• Our RDR and DCL Arabidopsis plants grew straight through mold at
times which leads us to believe that the genes that were missing in
these plants may have played a role in mold resistance.
• A few of the RDR and DCL plants exhibited leaf color changes from
green to purple as well. This leads us to believe that the triple
knockout mutant exhibits stress in plain agar. Those genes that were
knocked out play a role in leaf color and development.
Conclusion cont…
We found that a calcium nitrate concentration
.25 molar and higher would completely
suppress the plant’s development.
If we had been given more time we would have
kept using lower concentrations of the Calcium
Nitrate in order to see its effects on
Arabidopsis.
We’d like to thank…
• Dr. Lisa Johansen for helping us by supplying us
with seeds and soil, and for checking in with us to
see how our plants are growing.
• Mrs. Kristin Donley for helping us with our ideas
for how to grow our plants and for her overall
guidance.
Bibliography
• http//:www.jasons-indoor-guide-to-organic-andhydroponics-gardening.com; September 2008
• Andrew Fire and Craig Mellow, (2006) the Nobel prize in
physiology/medicine (http//:nobelprize.org)
• Elliot Meyerowitz, Prehistory and History of Arabidopsis
Research, Plant Physiology, January 2001, vol. 125, pp. 1519
• Sabina Leonelli, Arabidopsis, the Botanical Drosophilia:
from Mouse Cress to Model Organism; March 2007,
Endeavor, vol. 31 No. 1, pp.34-38
Bibliography
Franck Vazquez, Arabidopsis endogenous small RNAs: Highways and Byways, Trends
in Plant Science Vol. 11 No. 9 pp. 460-467.
Elliot M. Meyerowitz, Prehistory and History of Arabidopsis Research, Plant
Physiology, January 2001, Vol. 125 pp. 15-19.
Thomas Mitchell-Olds, Arabidopsis thaliana and its wild relatives: a model system for
ecology and evolution, Trends in Ecology and Evolution Vol. 16 No. 12 December
2001.
Sabina Leonelli, Arabidopsis, the botanical Drosophilia: from mouse cress to model
organism, Endeavor Vol 31 no.1, pp. 34-38.
Maria J. Clauss and Marcus A. Koch, poorly known relatives of Arabidopsis thaliana,
trends in Plant Science, vol. 11 No.9, pp.449-459.
Douglas C. Boyes, et al., Growth Stage-Based phenotypic analysis of Arabidopsis: A
Model for High Throughput Functional Genomics in Plants, The Plant Cell, Vol. 13,
1499-1510, July 2001.
Michael Wasenegger and Gabi Krczal, Nomenclature and functions of RNA-directed
RNA polymerases, Trends in Plant Science, Vol. 11 No. 3 pp.142-151, March 2006.
Fralin Biotechnology Center. “PREP Experiment Guide”. Virginia Polytechnic Institute
and State University. Dec. 1, 2008 http://www.prepbiotech.vt.edu/
Bibliography
• Andrew Fire and Craig Mello. “The Nobel Prize In
Physiology or Medicine for 2006.” NobelPrize.org.
November 20, 2008. http://nobelprize.org
• Fralin Biotechnology Center. “PREP Experiment
Guide”. Virginia Polytechnic Institute and State
University. Dec. 1, 2008
http://www.prepbiotech.vt.edu/