Functional validation of soybean root cell-type-specific promoters

Akash Nooka and Ian Meyer Author

08/03/2021 Added

46 Plays

Description

Research on root cell-type-specific promoters of soybeans

Searchable Transcript

Search:

[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.

The screen size you are trying to search captions on is too small!

You can always jump over to MediaHub and check it out there.

Comments

0 Comments

Functional validation of soybean root cell-type-specific promoters

Description

Searchable Transcript

Comments icon comment

Related Channels

Comments