Functional validation of soybean root cell-type-specific promoters

Akash Nooka and Ian Meyer Author

08/03/2021 Added

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Research on root cell-type-specific promoters of soybeans

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[00:00:00.150]The soybean, no,
[00:00:01.440]scientifically as glycine max is the most widely grown oil seed in the world.
[00:00:06.390]And its versatility's unparalleled in terms of usage from renewable
[00:00:11.190]fuels to animal feed,
[00:00:13.410]the bean has an extensive impact on humanity beyond its humble beginnings in the
[00:00:18.120]soil. However,
[00:00:20.160]there is still much information to be uncovered about glycine max.
[00:00:24.000]And in our research,
[00:00:25.860]we aim to reveal the specific use of its DNA within itself.
[00:00:30.480]Today I Akash Nuka and my colleague EMR bring to you our
[00:00:35.400]project tog functional validation of soybean root cell type specific
[00:00:39.750]promoters.
[00:00:40.940]In an organism.
[00:00:42.140]Each cell contains the entire blueprint of its species known as the genome
[00:00:46.640]constructed out of DNA, the language which transcends all organisms,
[00:00:50.840]the genome stores information necessary for species to develop and function
[00:00:54.620]properly. Nevertheless, the entirety of the genome,
[00:00:58.130]isn't all used at a cellular level. In our own bodies. The cells are eyes,
[00:01:02.930]skin, heart, and lungs all utilize our genome in different ways.
[00:01:07.220]In order to accomplish specific functions,
[00:01:09.560]the cells are capable of doing so by using one of the fundamental building
[00:01:12.950]blocks of the genome known as the gene. Similarly,
[00:01:16.190]soybeans are composed of a variety of cell types with distinct gene expressions,
[00:01:20.330]which are all driven by promoter sequences, regulatory parts of genes,
[00:01:24.080]which control the extent to which each gene is utilized by monitoring promoter
[00:01:28.610]activity. One can label genes to specific tissue types. In our research,
[00:01:33.170]we aim to take several highly expressed genes in glycine,
[00:01:36.320]max and map each one to a specific tissue type by observing the activity
[00:01:40.710]promoter sequences using molecular cellular and microscopic
[00:01:44.750]biotechnologies.
[00:01:47.800]Using a single cell approach.
[00:01:49.540]We identified 15 genes which demonstrated high specificity towards certain
[00:01:54.220]tissue types as seen in figure one figure one demonstrates the
[00:01:58.600]14,537 dots in the projection.
[00:02:01.900]They correspond to one nucleus.
[00:02:04.330]The relative distances represent the similarities each nuclear have in terms of
[00:02:08.620]gene expression,
[00:02:10.210]their color represents through cluster specificity by making use of an
[00:02:14.320]amplification technique known as PCR.
[00:02:16.900]We exponentially created a myriad of each promoter sequences from the 15 genes
[00:02:21.400]we analyzed in place of the sequences into a plasmid,
[00:02:24.490]which is a DNA ring that has been modified to last for antibiotic resistance,
[00:02:28.840]the implementation or sequences with done with the use of a recombination,
[00:02:33.520]a special enzyme that swaps a portion of DNA on the plasmid with our desired
[00:02:37.690]sequence as seen in figure two,
[00:02:40.390]the diagram above depicts our two recombinase reactions on both reactions.
[00:02:45.370]The clone is enzymes undergo the respective processes.
[00:02:49.230]After successful recombinase reaction,
[00:02:51.510]we were able to inter our plasmid into equal light by shocking the bacteria.
[00:02:56.010]The shock is capable of producing pores in the cell membrane,
[00:02:59.020]which allows plasmids to freely enter in a process known as electroporation.
[00:03:03.460]We can verify that our reaction was successful by growing our transformed
[00:03:06.940]bacteria and antibiotics,
[00:03:08.560]which they should now have resistance to with growth on antibiotic,
[00:03:12.700]Petri dishes and another PCR on the result in colonies,
[00:03:16.390]we can verify that our plasma has accessed. We've been recombined.
[00:03:20.230]Although the process seems simple.
[00:03:21.970]We encountered numerous challenges along our research process. For instance,
[00:03:26.680]our transformed bacteria demonstrated a limited amount of growth hindering our
[00:03:30.400]verification,
[00:03:32.200]making our job even more strenuous in obscene amount of false positive arose
[00:03:36.700]from our PCR verification altogether, these challenges combined,
[00:03:40.480]it created many setbacks.
[00:03:43.050]After verification of a successful recombination.
[00:03:46.200]We utilize another recombination transfer or target sequence directly downstream
[00:03:50.580]from reporter.
[00:03:51.960]The reporter will flag the activity of the promoter energy and of interest when
[00:03:55.980]they're being expressed within the roots as seen in figure three,
[00:03:59.430]after another round of phase two electroporation verification to ensure a new
[00:04:04.110]recombinants reaction when smoothly we're finally rates. In fact,
[00:04:07.590]glycine bags using Agrobacterium riser genes,
[00:04:11.070]which has a special capability of inserting its own DNA to its host.
[00:04:14.610]We injected young soybeans with the solution of transformed Agrobacterium right
[00:04:18.840]below the first Kylene where cells are constantly dividing for maximum
[00:04:22.650]efficiency left to grow for about eight days of brown cows forms
[00:04:27.840]from the infection site. Roots can be seen emerging,
[00:04:30.180]which are hopefully expressing a reporter slash promoter pair.
[00:04:33.990]We can verify their expression under the microscope here.
[00:04:37.350]We can see the cells of the soybean root specifically in blue ourselves would
[00:04:41.790]show activity in a reporter.
[00:04:44.310]Therefore they're expressing ourselves specific gene.
[00:04:47.610]Each gene is specific to a different cell type and therefore each reporter must
[00:04:52.500]be carefully observed to determine which specific cell type it is active in.
[00:04:57.270]Usually the entire process of extraction verification and transformation takes
[00:05:02.160]90 days to occur successfully.
[00:05:04.830]By validating cell type specificity of selected soybean plant promoter
[00:05:08.860]sequences.
[00:05:09.900]Our research project supports the idea that the creation of the transcript to me
[00:05:13.350]within a plan reveals the vast range of how the genomic DNA is used between
[00:05:17.220]cells. For instance, in figure four,
[00:05:20.100]we observed GFP fluorescence in soybean trickle blasts,
[00:05:23.580]which were driven by the promoter sequence activity of the Gleimer [inaudible]
[00:05:27.400]1 4 9 1 0 0 gene expressed in cluster 11 as seen in figure
[00:05:32.070]five. Furthermore,
[00:05:34.140]the result helps us functionally annotate this cluster as the trickle blast
[00:05:37.710]cluster.
[00:05:38.850]Our work also leads to the creation of a library of soybean root cell type
[00:05:43.350]specific promoters that will help foster new strategies in the field of
[00:05:47.190]synthetic biology.
[00:05:48.960]Ultimately it will facilitate the establishment of modern strategies to control
[00:05:53.340]and study the biology of specific cell types.

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Functional validation of soybean root cell-type-specific promoters

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