Earth ScienceEnvironment

DNA Analysis of the Sucrose Synthase Enzyme in Genetically Modified and Heirloom Pisum sativum

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Abstract:

The world’s changing demands for crops has resulted in the genetic modification of plants’ genotypes, to increase crop yield and resist invading bacteria. Sucrose synthase (SuSy) is an enzyme that synthesizes sucrose; sucrose is integral for plant growth and the formation of carbohydrates. Two varieties of pea plants, genetically modified Pisum sativum and heirloom Pisum sativum, were grown and tested for the presence of different SuSy genes. Whether the genetically modified variant of peas would have higher levels of sucrose than in the SuSy gene in the heirloom was explored and documented. The hypothesis tested three different genes: PsSus1, PsSus2, and PSUT. Data collection confirmed the presence of the mutated PsSus2 gene in the genetically modified variant and the PSUT gene in the heirloom variant. In addition, DNA sequencing showed taller, increased quality peaks of the gene tested in the genetically modified plant, indicating high concentrations of the sucrose synthase enzyme. The PsSus2 gene controls gene expression. Increased activity of SuSy in the PsSus2 gene signals the enzyme’s synthesis pathways more, which will then produce more sucrose. The results displayed that there is a difference in sucrose levels in different generations of Pisum sativum. Findings from the analysis can be further applied to the global demand for crops. Increased levels of sucrose synthase result in higher amounts of sucrose, which is beneficial for rapid plant growth.

Introduction:

Genetic modification is a practice that has been used conventionally for many years; certain traits are selected for and this process can help to diversify the gene pool. Genetically modified plants are resistant to certain diseases and change the metabolic processes in crop plants[1]. Current research has shown that genes involved in photosynthesis can speed up the rate of growth in a crop plant.[2] With the global population’s increase in size, the demand for crop yield has risen. The effects of genetic modification in certain crops and their genes can help to find a solution that meets today’s agricultural needs.

Sucrose synthase (SuSy) is an enzyme that produces sucrose and catalyzes plant metabolism and growth. It is a glucose-fructose disaccharide, and is transported throughout the plant in the phloem.[3] SuSy is involved in the reaction of NDP-glucose and D-fructose to yield sucrose and nucleoside-diphosphate kinase during photosynthesis.[4] Sucrose synthase synthesizes biological sucrose pathways and controls function within the plants’ seeds, stem, and leaves. In the plant Pisum sativum, sucrose synthase is most active within the roots. Elevated activity of sucrose synthase has been shown to increase the growth of plants, and the overproduction of cellulose, a carbohydrate that supports the plant’s cell wall.[5] In contrast, decreased levels of SuSy results in the crop’s susceptibility to certain diseases and anaerobic plant stress.[6] When the sucrose synthase enzyme is genetically modified to become more present, the plant has a stronger stem due to starch synthesis and will grow faster.[7] This would serve to be beneficial for the agricultural industries as growth can become efficient and fast, allowing for more production of different crops.

The plants tested were genetically modified Pisum sativum and heirloom Pisum sativum. The genetically modified Pisum sativum we used is resistant to pathogens and stress related diseases. The heirloom is not genetically modified and is the original form of the sugar snap pea plant. Pisum sativum is a species of plant that is known to have rapid starch synthesis, produced from sucrose pathways in the plant.[8] The plant has fast-acting metabolism, making it easier to genetically modify than other crop plants. There were variable expressions of SuSy in carrot, contributing to increasing sugar solubility and sucrose presence during root development.[9] In addition, there was decreased expression of some of the encoding genes for SuSy, and three different genes were found as well.[10] An experiment with a focus on nodulated peas and nitrogen fixation gathered results that genes related to SuSy were PsSus1, PsSus2, and PSUT. These three isoforms of SuSy have also been found to be expressed differently in both level and locations of the plant.[11] Similarly, in the sugarcane plant, 4 gene families of SuSy were identified.[12] The PsSus1 gene transports sucrose throughout the cells.[13] The PsSus2 gene controls gene expression involving sucrose synthase enzymes and allows for controlled concentrations of sucrose, fructose, and starch.[14] The PSUT gene is a plastidic sugar transporter. Plastidic sugar transporters have the role of controlling plant stress such as weather and diseases.[15] Gene expression is important in the field of genetic modification because researchers can alter whole biological pathways to increase signaling and produce more proteins and enzymes.[16]

Drawing from conclusions about the presence of sucrose synthase in several different plants, it was postulated that sucrose synthase exists in different genes in the heirloom and genetically modified versions of Pisum sativum. Using the information about genetically modified organisms, we investigated whether there will be increased or decreased sucrose synthase levels when the gene is mutated or absent.

Methods:

Growing Pisum sativum plants

In this experiment, two varieties of pea plants were used: genetically modified Pisum sativum and heirloom Pisum sativum (Figure 1). The plants were first potted with a nitrogen-based fertilizer. Both the pea plants were grown under the same conditions. They were given twelve hours of light per day for three weeks under a Grow-Light bulb. The crops were watered every three to four days with distilled water. Upon germination, DNA extraction was conducted.

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Figure 1: Plants during Germination

Heirloom and genetically modified Pisum sativum at 20 days of growth, prior to DNA extraction.

Extraction of DNA of Pisum Sativum

A buffer solution of distilled water, salt, and detergent was prepared for lysis. The buffer solution causes cells to break open, to lyse, and initiates DNA extraction. Each variant of Pisum sativum was ground in the lysis solution. An equivalent volume of isopropanol was added to each DNA solution in order to precipitate the DNA. The tube was centrifuged for one minute and then the DNA was purified with isopropanol. The tube was centrifuged again for one minute, and then the DNA solution was eluted with distilled water. The DNA was stored under conditions of -30°C.

Amplification of DNA to Respective Sucrose Synthase Genes

Afterwards, Polymerase Chain Reaction (PCR) was conducted in order to denature, anneal, and amplify the DNA with the genes. Six tubes were obtained for the 3 sets of primers, reverse and forward, and two different variants of plants. Primers are molecules which mark starting and ending points for synthesis of genetic material. Five μL of PCR Buffer, 2.5 μL of the 10 μM solution of the forward primer and reverse primer, 0.5 μL of the Taq DNA Polymerase, 2 μL of the dNTP mix, 0.85 μL of the DNA, and 7.65 μL of distilled water were added to each tube. This step was repeated for each set of primers and variants of Pisum sativum. The PCR was conducted in a thermocycler machine.

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Figure 2: PCR Set-Up

Figure shows two different trials of Polymerase Chain Reaction using the thermocycler. Temperatures for each step are listed alongside the step duration per each cycle.

Separation of DNA Fragments In Order to View Genes

Agarose and TBS buffer mixture was prepared for gel electrophoresis. The set gel was placed in a gel electrophoresis box and was filled three-fourths full with a TBS buffer. The PCR solutions were then thawed, and 5 µL of loading dye was added to each PCR tube. 15 µL of each solution, including the loading dye, were placed into each well. One well was loaded with the DNA ladder. The box was then plugged into a voltage machine at 140 voltages for 45 minutes with 15 minute increments. Once complete, the gel was then transferred to a petri dish filled with a diluted solution of ethidium bromide and distilled water (Figure 3). The petri dish containing the gel was stored at 4 °C overnight.

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Figure 3: Preparation for Visualization of Genes

Agarose gel containing separated fragments of DNA from gel electrophoresis is soaked in ethidium bromide (EtBr).

Determination of Sucrose Synthase Genes

The gels were visualized using a UV light in order to find which genes were present. After determining the genes present, the DNA in the gel was cut out using a scalpel and placed in an Eppendorf tube in order to purify. Three volumes of agarose dissolving buffer were added to each tube and then incubated at 55 °C in order to dissolve the gel. The gel solution was placed in a spin column, centrifuged until there was flow through, and it was discarded. 200 µL of DNA wash buffer was added to each column, centrifuged, and flow through was discarded. This step was repeated twice. 6 µL of DNA elution buffer was then added to each column and centrifuged. DNA was then stored at 4°C. The purified DNA samples were then sent for Sanger DNA sequencing.

Results:

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Figure 4: Presence of SuSy Genes in Gel Electrophoresis: Heirloom

Results from the visualization of heirloom Pisum sativum. *Indicated = Gene does not have accurate base length

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Figure 5: Presence of SuSy Genes in Gel Electrophoresis: Genetically Modified

Results from the visualization of genetically modified Pisum sativum.

Three trials of gel electrophoresis were conducted for optimal results. The 2nd trial of PCR conducted (Figure 2), was used for trials two and three of gel electrophoresis, which brought accurate results. The procedure was conducted to determine which genes are present in the heirloom and genetically modified versions of Pisum sativum. Once soaked in the EtBr solution, the visualization deduced that the heirloom variety has PSUT in its genotype two out of three trials. The PsSus2 gene was found in one out of three trials but was not consistent enough to confirm its presence (Figure 4). The genetically modified species was found to have the PsSus2 gene present in two out of three trials (Figure 5). PSUT gene was also found in one out of three trials but was not consistent enough to confirm its presence. The PsSus1 gene was found in neither gene.

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Figure 6: DNA Sequences

The data collected on the PsSus2 gene is from the genetically modified plant and the PSUT gene from the heirloom. The middle column lists the expected gene sequences of the PsSus2 and PSUT genes.[17] The column to the right states the gene sequences we have collected in the experiment. Our PsSus2 gene sequence is altered, as expected. The PSUT gene sequence recorded from the heirloom is precisely similar to the expected gene sequence.

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Figure 7: Expected DNA Fragment Size v.s Experimental

The PSUT gene for the heirloom variant and the PsSus2 gene for the genetically modified variant are within their expected ranges.[18]

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Figure 8: Part of the Chromatogram of the PSUT Gene In Heirloom Pisum sativum

This showed peaks with quality levels primarily between 10 and 24. It has 178 bases of cytosine and guanine, and 228 bases of adenine and thymine.

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Figure 9: Part of the Chromatogram of Genetically Modified Pisum sativum

The results showed peaks with quality levels primarily between 10 and 37. It was found to have 199 bases of cytosine and guanine, and 196 bases of adenine and thymine.

From the sequencing results, it was found that the heirloom sequence for the PSUT gene varies from the genetically modified Pisum sativum sequence. The genetically modified sequence had more bases of cytosine and guanine than the heirloom. The heirloom sequence had more bases of adenine and thymine in contrast to the genetically modified plant. Overall, the chromatogram showed that the genetically modified Pisum sativum had a greater quantity of higher quality peaks than the heirloom.

Conclusion:

The results revealed, as hypothesized, that the genetically modified Pisum sativum has increased levels of sucrose synthase due to high concentrations of the SuSy gene. The high concentrations of the enzyme and the mutated gene sequence indicated an overactive presence of the gene in the crop. Thus it was concluded that the genetic modification of the PsSus2 gene will produce more sucrose synthase enzymes, and in turn, sucrose. The data collected from this research can be of advantage to the exponentially increasing population. As there is greater demand for crops, the data can help create methods to increase crop yield.

Discussion:

Higher quality peaks show an increased concentration and presence of the sucrose synthase enzyme in the genetically modified crop (Figure 9). It suggests that the amplified DNA strands bound to its respective gene more efficiently. The PSUT gene was present in one of the gel electrophoresis trials for the genetically modified version. However, the base length from the gel electrophoresis results was not visualized to be in the correct range of base numbers for the PSUT gene. Thus, it was concluded that the primers may not have been able to anneal correctly: attachment to the DNA template was incomplete, leading to missing base pairs and thus not exact base length. Annealment was not as effective as the PsSus2 gene results. From the visualization and sequencing results, the genetically modified Pisum sativum has a mutated gene sequence (Figure 6) and high concentrations of the sucrose synthase enzyme, as evidenced by high peaks in Figure 8. The gene that is found on multiple accounts in the genetically modified crop is the PsSus2 gene, which controls gene expression. This is useful because gene expression allows genetic modification to be easier and more effective for researchers.[19] When the gene expression is mutated, the signals sent within the gene are processed to create more of a certain enzyme.[20] In this case, it would mean that the over-expressed PsSus2 gene sends more signals to create the sucrose synthase enzymes.

Since the PsSus2 gene has high activity in the genetically modified variant, this is of knowledge to the genetic modification of sucrose synthase. An increase of sucrose synthase in the PsSus2 gene means that this gene can be pinpointed and modified to produce more sucrose enzymes. By editing the PsSus2 gene, the concentrations of sucrose yielded will become higher, and the pathways altered.

It was found that the heirloom Pisum Sativum did not fully bind to the PsSus2 gene since the base pair lengths do not match up to the expected range (Figure 7). The PSUT gene which is present in the heirloom was not overexpressed. This is because the gene sequence was not mutated (Figure 7) and the quality levels of the peaks were also not as high as the genetically modified, which shows that the genes did not fully completely bind to the DNA during PCR. It should be noted however, the heirloom had the PsSus2 gene and the genetically modified variant had the PSUT gene somewhat present, but the results from visualization were not consistent and the base pair lengths were not in the correct ranges, thus it was not accounted for.

The results support the hypothesis – the genetically modified variant of Pisum sativum had a higher sucrose synthase concentration. As mentioned before, the PsSus2 gene was altered, and since it is the gene responsible for gene expression, in this situation it indicates that the genetically modified Pisum sativum, has more pathways producing the SuSy enzymes. The data provided shows that mutation of the PsSus2 gene in genetically modified Pisum sativum results in higher concentrations of SuSy, which will therefore produce more sucrose.

Since the mutation of the PsSus2 gene in the genetically modified Pisum sativum shows higher concentrations of the sucrose synthase enzyme, this information can potentially be used further to genetically modify other crop plants to have higher sucrose levels for rapid crop growth.

Acknowledgments:

We would like to thank our advisor, Soumya Suresh for guiding our project and devoting her time to us. In addition, we would like to thank The Center For Advanced Study and Olive Children Foundation for providing us the lab space and fundings to conduct the research in this project.

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Biographies:

Simone Khandpekar is currently a junior in Irvington High School in Fremont, CA. Simone has previously done research on NNRTIs and HIV, and cellular aging. Simone enjoys writing poetry and is a published poet and artist as well. She is also a certified yoga teacher and practices yoga. She hopes to pursue her dream as being a part of the medical field someday.

 

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Anushka Rajasekhar is fifteen years old and attends Amador Valley High School in Pleasanton, California. Anushka enjoys learning how today’s most pressing issues can be solved with scientific research. She finds topics in public health and infectious diseases interesting, and is currently doing research in organic chemistry. Outside of science, she enjoys extreme hiking, badminton, plays the piano, and volunteers with several humanitarian aid societies.

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Shloka Raghavan is fifteen years old and attends Amador Valley High School in Pleasanton, CA. Shloka enjoys learning about biomedical research especially pertaining to the brain. She has previously done research in the psychology field relating depression in adolescents. Shloka is currently doing research in organic chemistry. She practices yoga and EFT and enjoys running and playing both eastern and western violin.

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