lab report microrna + nutrients final draft
TRANSCRIPT
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Nicole Celani Biol 220/ Section 012T.A. Laura Bennett 3/6/2012
Title:
Investigating microRNA expression by varying nutrient concentration available to Arabidopsis
thaliana
Introduction:
Arabidopsis thaliana is the model organism chosen for investigation of miRNA
expression because its entire genome has been sequenced (Weems 362–369). This organism also
has a short life cycle and grows relatively quick with restricted space and this also makes it
useful for lab research (Weems 362–369). The nutrient concentrations that were varied in this
study included phosphorus and sulfur. These nutrients are specifically varied because they are
considered macronutrients that are required for plant growth (Axtell). Phosphorus is a
component of nucleic acids, phospholipids, ATP and coenzyme. Sulfur is also a component of
coenzymes as well as proteins (Axtell). These are important nutrients for research because
macronutrient such as sulfur and phosphorus are limiting for plant growth and are often added
via fertilizers to soils (Axtell). Plants respond to varying levels of nutrient concentrations by
controlling their gene expression. Why waste energy creating proteins involving sulfur when
there is no sulfur available to the plant? To prevent energy loss, plants control gene expression
via miRNA. miRNA degrades mRNA that is creating un-useful proteins. The function of
miRNA is to bind to single stranded mRNA creating double stranded RNA. The plant
recognizes the double stranded mRNA as foreign and breaks down the complex. This acts as a
negative post-transcriptional control (Axtell). The four microRNAs used for this investigation
were miR156, miR395, miR399 and miR398. The developmental regulator, miR156, has no
nutrient requirement and would be expected to show no change due to nutrient levels changing
(Hsieh 2120-2132). miR399 is up-regulated during phosphate starvation and thus targets genes
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Nicole Celani Biol 220/ Section 012T.A. Laura Bennett 3/6/2012
in phosphorus uptake and metabolism (Hsieh 2120-2132). miR398 is regulated when there is
Copper/Zinc starvation, targeting the enzyme copper/zinc superoxide dismutases (Bouché 684–
686). This stress responsive miRNA has low levels of expression in both low sulfur and
phosphorus environments (Bouché 684–686). Lastly, miR395 depends on sulfur concentrations
and targets mRNAs involved in sulfur metabolisms (Hsieh 2120-2132). These miRNAs should
show different effects due to the nutrients limitations and this is why they were chosen.
The purpose of this study is to examine differential gene expression under low nutrient
levels of sulfur and phosphorus. The procedure for this experiment was to plate Arabidopsis
thaliana on three mediums including full media, low phosphorus and no sulfur. The miRNAs
were then extracted from seedlings using a Qiagen kit. The data was then analyzed using qt-
PCR analysis.
Results:
Data was collected for three graphs throughout this investigation. Figure 1, 2 and 3
represents the normalized relative accumulations for miR156, miR398, miR399 and miR395
under low phosphorus and low sulfur conditions compared to the full media plate. Figure 1, 2
and 3 represent data from the previous studies data, section 012’s data and data collected from
the entire 240 class, respectively. In all three replicates of the investigation miR156 showed very
little to no up regulation. All data sets show a very low expression level of miR156 as seen in
figures 1, 2 and 3. However, the section data shows a slightly higher level of expression under
low phosphorus conditions compared the previous study and the class data. miR395 showed
extreme up regulation across all three replicates under low sulfur conditions. Under low
phosphorus conditions miR395 showed little up regulation in the section data and down
regulation in the previous studies data and the class 240’s data. miR395 showed the strongest
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Nicole Celani Biol 220/ Section 012T.A. Laura Bennett 3/6/2012
down regulation in the previous study’s data under low phosphorus conditions compared to the
other sample sets. miR398 showed repressed expression in both low sulfur and low phosphorus
conditions across all three sample sets. As seen in Figure 1, the section data showed down
regulation of miR398 in both conditions and more strongly under low phosphorus. Figure 3
shows that the class data has both conditions expressing down regulation but more strongly
under low sulfur conditions. However, the section data shows low levels of up regulation of
miR398 during low phosphorus conditions compared to the previous study and overall class data.
Lastly, miR399 showed higher levels of expression under low phosphorus conditions across all
three sample sets. All three figures also correlate in low levels of miR399 expression during low
sulfur conditions. The class data showed the strongest down regulation under low sulfur
conditions for miR399, as seen in figure 3. The experimental results obtained for my performed
research in wells C7-C12, for low phosphorus conditions and miR395 came back as faulty
results.
Discussion:
Differential gene expression under low phosphorus and low sulfur conditions for each
miRNA had different responses to the nutrient concentrations as was expected. miR156 was
expected not to have been regulated by nutrient suppression because it is not involved in
phosphorus or sulfur processes (Hsieh 2120-2132). This corresponds well to all three graphs, in
that there are low levels of expression in all data sets for both nutrient deficiencies. This shows
that miRNAs are specific to target certain functional machinery for degradation and regulation.
miR395 showed extreme up regulation of expression across all three sample sets for low sulfur
conditions. This correlated exactly with what would be expected considering the function of
miR395 is to target mRNAs involved in sulfur metabolism when sulfur is not present (Hsieh
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Nicole Celani Biol 220/ Section 012T.A. Laura Bennett 3/6/2012
2120-2132). miR395 was not expressed or had down regulation under low phosphorus
conditions because it does not function in the degradation of phosphorus machinery and this is
also supported by figures 1, 2 and 3. When considering miR398 the graph displays low levels of
expression across all three data sets. Knowing the expression of miR398 is to be repressed under
low sulfur and phosphorus conditions the graphs support the observation of low expression
levels (Bouché 684–686). The cell is preventing the breakdown of CuZn-oxide dismutaes by
suppressing the formation of miR398 (Bouché 684–686). However, the section data did shows a
slight up regulation of miR398 compared to the class and previous study’s data, as seen in
figures 1, 2 and 3. Lastly, miR399 was up regulated under low phosphorus conditions compared
to low sulfur conditions across the data sets. miR399’s function is to cause the degradation of
machinery involved in phosphorus uptake and metabolism under low phosphorus conditions to
conserve energy (Hsieh 2120-2132). This is reflected in figures 1, 2 and 3. miR399 was not up-
regulated in low sulfur conditions because degradation of this specific machinery is not required.
On a full media plate all usual machinery and miRNAs were present in natural concentrations
when all required nutrients were present.
When considering all three data sets it seemed that the collection of all 33 sections in the
class 240 data had the best correlation to the functions of each miRNA. This is most likely due
to the large set of replicates collected for this graph compared to the other two data sets. Sources
of error may have occurred in many steps along the procedure. I specifically performed a low
phosphorus experiment in wells C7-C12 using miRNA primer 395 which resulted in faulty data.
These non-normal values may have been due to errors in the procedure. This may have been due
to lack of starting tissue in low phosphorus mediums because the growth was very limited.
Errors may have occurred during RNA extraction, PCR amplification and human error. Also,
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Nicole Celani Biol 220/ Section 012T.A. Laura Bennett 3/6/2012
introduction of RNases may have contaminated samples during RNA extraction and not
thoroughly grinding plant tissue may have also produced faulty data.
This research is important for the relative sulfur and phosphorus conditions in soils used
for farming and how this is effecting plant growth in agriculture. These macronutrients have
dramatic effects on plant growth and this research establishes how these nutrients affect the
expression of miRNA and thus growth. This research also provides insight to the functions of
the microRNA involved in specific nutrient deficiencies. This research leads to further
investigation of the effects of adding sulfur and phosphorus in large quantities to mediums
instead of low concentrations. Measuring the results of different levels of miRNA expression
and growth rates would be helpful information in optimizing plant growth. This would be
useful in understanding the exact concentrations of macronutrients that plants require for
growing at optimal rates. Particularly, looking more closely at miR398 would be interesting
because it had experienced down regulation. This would help in understanding what miR398’s
function is when phosphorus and sulfur nutrients are present. Observing different microRNAs
that regulate gene expression help to understand nutrient requirements in plants.
Materials and Methods:
Arabidopsis thaliana seeds were plated on three different media types including low
phosphorus, low sulfur and a full media plates. The control in this experiment was the full media
plate. The independent variable manipulated in this study was the media type that the seeds were
grown on. The dependent variable measured was the varying levels of miRNA expression. My
lab partner and I did a low phosphorus plate. The plates were then allowed to grow for two
weeks. The full media plate was used in comparison to the low phosphorus and low sulfur plates.
After growth the plants were collected with sterilized tweezers and grinded using a lysis mix and
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Nicole Celani Biol 220/ Section 012T.A. Laura Bennett 3/6/2012
a pestle. Next, the kit used for isolating the small RNAs was a Qiagen kit following the
manufacturer’s protocol. The microRNAs were then run in the thermocycler performing reverse
transcription reactions to convert the RNAs into single-stranded complementary DNAs.
Reverse Transcriptase Master Mix was used for the reverse transcription reactions. Lastly,
quantitative real-time polymerase chain reaction (qt-PCR) was used to analyze the microRNA
levels. Four microRNAs were examined for each media type. Every media type was analyzed
using this procedure and U6 was used as the reference gene for analysis. A U6 Master mix and
miRNA master mix were used for analysis of each media type. The four primers used for
analysis were miR156, miR395, miR398 and miR399. My lab partner and I used miRNA primer
395. The efficiency values, E, were then obtained from the previous study (data shown in figure
1) and was not actually calculated. From this information the cycle threshold, Ct, values were
calculated and used to calculate the normalized relative abundances of each miRNA. These
abundances were then used for analysis of the varying levels of miRNA expression in low sulfur
and low phosphors conditions.
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Nicole Celani Biol 220/ Section 012T.A. Laura Bennett 3/6/2012
Figures: All blue bars are representations of median RAn values of miRNAs in low
phosphorus conditions and all red bars are median RAn values of miRNAs in low sulfur
conditions.
Figure 1: The bar graph is a representation
of the normalized relative accumulation
values for the four miRNAs for a previous
study. The graph compares the regulation in
both low sulfur and low phosphorus
conditions.
Figure 2: The bar graph is a representation
of the normalized relative accumulation
values for the four miRNAs for Section
012’s Data. The graph compares the
regulation in both low sulfur and low
phosphorus conditions.
Figure 3: The bar graph is a representation
of the normalized relative accumulation values for the four miRNAs for all 33 sections of class
240’s data. The graph compares the regulation in both low sulfur and low phosphorus
conditions.
miR156 miR395 miR398 miR3990.01
0.1
1
10
100
1000
LowPLowS
The median RAn values of a Previous Study’s Data
The median RAn values of Section 012 Data
The median RAn values of Class 240’s Data
miR156 miR395 miR398 miR3991
10
100
1000
10000
LowPLowS
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Nicole Celani Biol 220/ Section 012T.A. Laura Bennett 3/6/2012
References:
Bouché, Nicolas. "New insights into miR398 functions in
Arabidopsis." Plant Signal Behav.. 5.6 (2010): 684–
686. Web. 6 Mar. 2012.
<http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3001559/?tool=pmcentrez>.
Hsieh, Li-Ching, Shu-I Lin, Arthur Chun-Chieh Shih, June-Wei Chen, and Wei-Yi Lin.
"Uncovering Small RNA-Mediated Responses to Phosphate Deficiency in Arabidopsis
by Deep Sequencing." Plant Physiol.. 151.4 (2009 ): 2120–2132. Web. 6 Mar. 2012.
<http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2785986/>.
“microRNA and Plant Nutrition” Axtell, M. Burpee, D. , and Nelson, K. Department of Biology,
The Pennsylvania State University, University Park, PA. (2012)
Weems, Danforth, Neil Miller, Margarita Garcia-Hernandez, Eva Huala, and Seung Rhee.
"Design, Implementation and Maintenance of a Model Organism Database for
Arabidopsis thaliana ." Comp Funct Genomics. 5.4 (2004): 362–369. Web.
<http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2447457/>.
miR156 miR395 miR398 miR3990.01
0.1
1
10
100
1000
10000
LowPLowS