Title
Occurrence of microRNA expressions using two macronutrient deficiencies presented to Arabidopsis Thaliana
Introduction
Arabidopsis Thaliana was the model chosen for this experiment. It was used for miRNA expression because its entire genome is already sequenced (Weems 362-369). It is also easy to grow in difficult conditions and has a short lifespan. This all makes it an easy plant to work with for research (Weems 362-369). The macronutrients phosphorus and sulfur were used for the study because they are essential for plant growth (Axtell). Both phosphorus and sulfur are components of coenzymes and proteins (Axtell). Controlling gene expression via miRNA is the plant’s way of responding to limiting levels of these essential macronutrients. miRNA works by binding to single stranded mRNA which creates double stranded RNA. Thus the plant identifies the new double stranded mRNA as inferior and attacks it.
For the experiment, there were four microRNAs used including miR156, miR395, miR398, and miR399. miR156 was the control and has no nutrient requirement so it would be expected to not show any changes in nutrient levels (Hsieh 2120-2132). miR395 depends on sulfur concentrations and targets mRNAs involved in sulfur metabolisms (Hsieh 2120-2132). miR398 is regulated when there is Copper/Zinc starvation, targeting the enzyme copper/zinc superoxide dismutase (Bouché 684– 686). miR399 is up-regulated during phosphate starvation and thus targets phosphorus uptake and metabolism (Hsieh 2120- 2132). This stress responsive miRNA has low levels of expression in both low sulfur and phosphorus environments (Bouché 684- 686). The purpose of this experiment was to examine how gene expression in Arabidopsis Thaliana is affected under deficient nutrient levels of sulfur and phosphorus. Materials and Methods Arabidopsis Thaliana was to be placed on three mediums; full media, low phosphorus, and no sulfur and left to grow. …show more content…
Then the miRNAs would be extracted from each of the three mediums using a Sigma mirPremier miRNA isolation kit and then analyzed using qt-PCR. The control used was the full media plate. The media type that the seeds were grown on was the independent variable in the experiment while the dependent variable was the different levels of miRNA expression. The plants were then collected into a tube and grinded in a lysis mix with a pestle. To isolate the small RNAs, the Sigma mirPremier miRNA isolation kit was used. Reverse Transcriptase Master Mix was then used for the reverse transcription reactions by putting the miRNAs in a thermocycler to convert them into single stranded DNA. Finally, qt-PCR was used to analyze the levels of miRNA. Each media type had four miRNAs designated and all media types were analyzed using a U6 Master Mix and miRNA Master Mix. The four miRNA primers obtained from each media type were miR156, miR395, miR398 and miR399. The efficiency values (E) were obtained from a previous miRNA study done with given media type values. From these values, the cycle threshold values (Ct) were calculated and were then used to calculate the normalized relative abundance values (RAn) of each miRNA. From these calculations, the varying levels of miRNA expression in low sulfur and low phosphorus conditions were found. Results After the RAn values were calculated, a graph was created representing those values for miRNA 156, miRNA 395, miRNA 398, and miRNA 399 subjected to low phosphorus and no
Occurrence of microRNA expressions using two macronutrient deficiencies presented to Arabidopsis Thaliana
Introduction
Arabidopsis Thaliana was the model chosen for this experiment. It was used for miRNA expression because its entire genome is already sequenced (Weems 362-369). It is also easy to grow in difficult conditions and has a short lifespan. This all makes it an easy plant to work with for research (Weems 362-369). The macronutrients phosphorus and sulfur were used for the study because they are essential for plant growth (Axtell). Both phosphorus and sulfur are components of coenzymes and proteins (Axtell). Controlling gene expression via miRNA is the plant’s way of responding to limiting levels of these essential macronutrients. miRNA works by binding to single stranded mRNA which creates double stranded RNA. Thus the plant identifies the new double stranded mRNA as inferior and attacks it.
For the experiment, there were four microRNAs used including miR156, miR395, miR398, and miR399. miR156 was the control and has no nutrient requirement so it would be expected to not show any changes in nutrient levels (Hsieh 2120-2132). miR395 depends on sulfur concentrations and targets mRNAs involved in sulfur metabolisms (Hsieh 2120-2132). miR398 is regulated when there is Copper/Zinc starvation, targeting the enzyme copper/zinc superoxide dismutase (Bouché 684– 686). miR399 is up-regulated during phosphate starvation and thus targets phosphorus uptake and metabolism (Hsieh 2120- 2132). This stress responsive miRNA has low levels of expression in both low sulfur and phosphorus environments (Bouché 684- 686). The purpose of this experiment was to examine how gene expression in Arabidopsis Thaliana is affected under deficient nutrient levels of sulfur and phosphorus. Materials and Methods Arabidopsis Thaliana was to be placed on three mediums; full media, low phosphorus, and no sulfur and left to grow. …show more content…
Then the miRNAs would be extracted from each of the three mediums using a Sigma mirPremier miRNA isolation kit and then analyzed using qt-PCR. The control used was the full media plate. The media type that the seeds were grown on was the independent variable in the experiment while the dependent variable was the different levels of miRNA expression. The plants were then collected into a tube and grinded in a lysis mix with a pestle. To isolate the small RNAs, the Sigma mirPremier miRNA isolation kit was used. Reverse Transcriptase Master Mix was then used for the reverse transcription reactions by putting the miRNAs in a thermocycler to convert them into single stranded DNA. Finally, qt-PCR was used to analyze the levels of miRNA. Each media type had four miRNAs designated and all media types were analyzed using a U6 Master Mix and miRNA Master Mix. The four miRNA primers obtained from each media type were miR156, miR395, miR398 and miR399. The efficiency values (E) were obtained from a previous miRNA study done with given media type values. From these values, the cycle threshold values (Ct) were calculated and were then used to calculate the normalized relative abundance values (RAn) of each miRNA. From these calculations, the varying levels of miRNA expression in low sulfur and low phosphorus conditions were found. Results After the RAn values were calculated, a graph was created representing those values for miRNA 156, miRNA 395, miRNA 398, and miRNA 399 subjected to low phosphorus and no