Elucidating Gene Expression Patterns for Resilient Maize Lines Under Nitrogen Stress

Alice Guo Author

08/03/2021 Added

29 Plays

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[00:00:00.630]Hello. My name is Alice Guo and this summer,
[00:00:03.000]my research consisted of elucidating gene expression patterns for resilient maize
[00:00:07.170]lines under nitrogen stress supervised by Dr. Brandi Sigmon. Crops
[00:00:11.670]such as maize have varied resilience to environmental factors that contribute to
[00:00:15.540]a biotic stress such as nitrogen deficiency.
[00:00:18.550]Nitrogen plays a major role in the proper growth and development of maize and is
[00:00:22.380]essential to crop production,
[00:00:23.970]being the most critical nutrient for achieving optimal crop yield.
[00:00:27.570]Changing the amount of nitrogen can also directly impact physiological changes
[00:00:31.650]in the crop that ultimately affect yield. From our previous experiments,
[00:00:35.490]we found nitrogen stressed ears that are shorter,
[00:00:37.770]have incomplete fill, aborted kernels and have smaller tassels with fewer branches
[00:00:42.750]as shown in photo
[00:00:43.710]B as compared to A. The photo in the bottom right corner shows how overall
[00:00:48.660]ears under nitrogen stress were shorter than their unstressed counterparts.
[00:00:53.220]In previous field and greenhouse studies,
[00:00:55.350]we have found maize lines that differ and their resiliency to nitrogen stress.
[00:00:59.550]The most consistently resilient lines of maize have been identified as the lines
[00:01:03.150]A619 and A661.
[00:01:05.850]While the non resilient lines of maize have been identified as the lines NC368
[00:01:10.320]and B84. However,
[00:01:12.240]we still do not know why these specific lines are more resilient and perform
[00:01:16.290]better under nitrogen stress than the non resilient lines.
[00:01:20.280]To identify the potential genomic factors associated with the differences in
[00:01:24.330]nitrogen deficiency resilience,
[00:01:26.850]we conducted Genome-Wide Association Studies, also known as GWAS. From
[00:01:31.770]these analyses,
[00:01:32.520]we now have a list of candidate genes that will allow us to better understand
[00:01:36.300]their role in abiotic stress response. In this study,
[00:01:40.200]I aimed to verify targets for breeding more nitrogen resilient maize.
[00:01:44.340]This can aid in breeding efforts to reduce the amount of nitrogen inputs for
[00:01:47.910]farmers,
[00:01:48.390]which will be both cost efficient and better for the environment. To do this.
[00:01:52.770]I developed gene expression profiles for the candidate genes identified from GWAS
[00:01:57.330]in order to better understand the possible role of these genes in response to
[00:02:00.960]nitrogen stress throughout plant development.
[00:02:04.730]I grew both nitrogen stress resilient and non-resilient maize lines in the
[00:02:09.500]greenhouse and treated these with both Hoagland full nutrient solution
[00:02:14.180]and Hoagland solution prepared with only 20% nitrogen rather than 100%
[00:02:18.740]nitrogen.
[00:02:20.060]During this time, I developed and tested primers for eight total candidate genes
[00:02:24.230]previously identified from GWAS.
[00:02:26.420]These include three genes hypothesized to be expressed in inflorescences and
[00:02:30.530]five genes hypothesized to be expressed in roots.
[00:02:34.250]These genes were previous identified in GWAS results from maize inflorescence
[00:02:38.180]traits and their plasticity under nitrogen stress. In addition,
[00:02:42.230]I harvested tissues from the leaf, root and shoot apical meristems, and developing
[00:02:47.030]inflorescences at various developmental time points.
[00:02:51.050]Here's a photo exhibiting the effects of nitrogen stress with the minus nitrogen
[00:02:55.580]plant showing signs of stress. Following the tissue collection,
[00:03:00.400]I performed RNA extractions on the harvested tissues and ran a reverse
[00:03:04.840]transcriptase polymerase chain reaction,
[00:03:07.390]also known as RT-PCR, as seen in this photo, on the extraction
[00:03:12.310]RNA samples using the primers I had developed. From the results of
[00:03:17.290]the RT-PCR, I'll be able to observe varying levels of gene expression from the
[00:03:21.730]electrophoresis gels that will allow me to compare the data that is relative to
[00:03:25.810]each other, giving semi quantitative data.
[00:03:30.130]During my plant dissections,
[00:03:31.540]I harvested leaf, root, and shoot apical meristem tissues from the four lines of
[00:03:36.070]maze during the V4 stage of development.
[00:03:39.280]I collected tissues for the developing tassels at stages
[00:03:42.790]V6 and V8. For the ears, I collected tissues
[00:03:47.530]during the developmental stages of V8, V10, V11,
[00:03:52.330]V12 and V13.
[00:03:55.210]The eight candidate genes that were chosen from previous Genome-Wide
[00:03:59.680]Association Studies are shown in the table above. These include three genes
[00:04:04.570]hypothesized to be expressed in inflorescences highlighted in yellow and five
[00:04:09.490]genes hypothesized to be expressed in roots,
[00:04:11.500]which are highlighted in red. With this information,
[00:04:13.960]I was able to develop and test primers for each of the genes pictured above.
[00:04:18.010]This here is a figure exhibiting expression data associated with this gene.
[00:04:22.840]I chose this gene because of its strong expression in root tissues as shown
[00:04:26.650]here. In order to design my primer,
[00:04:31.150]I retrieved sequences for transcripts of genes and identified the forward and
[00:04:34.720]reverse sequences in the exons of the sequence.
[00:04:37.900]Here's an example of a gene transcript from the gene from the previous slide.
[00:04:43.390]The forward and reverse sequences are shown here in bold,
[00:04:46.270]and the exons are highlighted in blue. Using this information,
[00:04:49.840]I tested the primer on DNA and ran a polymerase chain reaction before doing the
[00:04:54.070]RT-PCR.
[00:04:55.660]This gel electrophoresis image is a result of a PCR
[00:05:00.430]I tested using the primer shown above. Due to the strong expression,
[00:05:04.570]I later ran an RT-PCR using this primer.
[00:05:09.520]The results from the RT-PCR were inconclusive and could be attributed to three
[00:05:13.630]possibilities.
[00:05:14.890]One being that the RNA had degraded, a second possibility being that the gene is
[00:05:19.210]not expressed in the tissues I had harvested and a third possibility being that
[00:05:23.260]the RTP-CR cycling conditions were off. For example,
[00:05:27.670]the annealing temperature could be too high or the extension time was too short.
[00:05:31.870]I have been troubleshooting to figure out what made my results inconclusive. In
[00:05:35.830]doing so,
[00:05:36.370]I obtained a control actin primer known to exhibit strong expression in many
[00:05:41.260]tissues to narrow down the possibilities.
[00:05:43.930]This electrophoresis gel is the result of an RT-PCR that I ran to test my RNA
[00:05:48.700]samples for degradation. The results of this gel tell me that the RNA in fact,
[00:05:53.200]did not degrade, and tht I can potentially move forward with testing
[00:05:56.020]other primers in the future.
[00:05:59.410]Here are figures to show how the nitrogen stress affected the four lines during
[00:06:04.120]various stages of maize development. On the bottom left,
[00:06:07.510]it is clear that the tassels were much larger in lines
[00:06:10.870]A619 and A661 compared to B84 and
[00:06:15.490]NC368. Additionally, within both of the A lines themselves,
[00:06:20.230]the minus nitrogens tassels are smaller than its plus nitrogen counterpart
[00:06:24.100]providing further evidence of the effects of nitrogen deficiency. In the figure
[00:06:28.900]on the top right, the A lines have larger ears across the board.
[00:06:35.230]The photo in the top left corner was a photo taken of a developing tassel during
[00:06:39.490]tissue dissections, and is a nice visual that shows the size of a tassel.
[00:06:44.200]It is possible that the A lines use nitrogen earlier in flowering tissue
[00:06:48.580]development compared to NC368 and B84. The latter lines may be
[00:06:53.560]developing other tissues and deplete the nitrogen source before it comes
[00:06:57.790]time for them to flower.
[00:07:00.760]Nitrogen has proven to be essential for the proper growth and development of
[00:07:04.240]maize, but nitrogen fertilizer is costly.
[00:07:06.910]Although innovative solutions have been brought forward through research,
[00:07:10.150]there's still much to be discovered and improved upon. By gaining an
[00:07:13.570]understanding of the different factors that affect a nitrogen deficiency in
[00:07:17.080]plant development,
[00:07:18.490]this may contribute to breeding efforts in enhancing crop production while
[00:07:22.420]accounting for costs and environmental effects.
[00:07:25.360]I've gained a better understanding of the effects of nitrogen deficiency in
[00:07:28.780]plant development and helped narrow down the list of candidate genes for
[00:07:32.650]potential future CRISPR/Cas9 or other knockout experiments.
[00:07:37.120]I plan to continue researching the effects of nitrogen stress on various maize
[00:07:41.230]lines and hope to further elucidate,
[00:07:43.960]further elucidate gene expression patterns for different primers. Additionally,
[00:07:48.220]I would like to work on a RNA sequencing project with the extra maize tissues I
[00:07:52.330]had gathered during harvesting. I'd like to thank my supervisor, Dr.
[00:07:57.100]Brandi Sigmon for her guidance and support, Jon Turkus and the Schnable lab for
[00:08:01.960]aid in my RNA extraction process,
[00:08:04.330]Mackenzie Zwiener for aid during my plant dissections and RT-PCR process,
[00:08:09.400]as well as CRRI for support during my time as an undergrad
[00:08:14.050]researcher. And lastly, UCARE for funding this summer research project.

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Elucidating Gene Expression Patterns for Resilient Maize Lines Under Nitrogen Stress

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