Functional validation of soybean root cell-type-specific promoters
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
- [00:00:40.940]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
- [00:01:47.800]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
- [00:03:32.200]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
- [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
- [00:05:09.900]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
- [00:05:38.850]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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