1. Put Your Dukes Up AT5G03220! Studying Embryo Lethality of
Knockout T-DNA Mutagenesis on
Arabidopsis Thaliana Genes
By Aditi S. Hendi

2. What was studied of AT5G03220?
Physical Characteristics and Descriptive Analysis of Gene
Determination of T-DNA Insertion Candidates for Knocking Out the Gene
Activity at Various Stages in Plant Development of Gene
Genotyping Plants Affected or Exposed to T-DNA
Effect of Results of Genotyping

3. What is AT5G03220? Located on Arabidopsis Chromosome 5
Reverse Complementary Oriented
Has 6 exons, 5 introns, 2 5’ UTRs, & 1 3’ UTR
AT5G03210 is 4167 bp downstream and AT5G03230 is 656 bp upstream to it

4. What else is known about AT5G03220?
Codes for transcriptional co-activator or related gene
Contains the F-box motif on the amino-terminus
Works in conjunction with other proteins in the regulation of transcription

5. How does Scarlet Runner Bean model mRNA activity of Arabidopsis?
Approximations of activity levels of ortholog SRB gene was used to model those expected of Arabidopsis (right)
The mRNA of the gene was found to be inactive, or present in trace amounts, in the flower organs of SRB
However it was only an approximation…

6. When is AT5G03220 active in Arabidopsis Thaliana?
Mutant line results indicate more up-regulation post-24-Hr seed stage, and more down-regulation slightly after 30 days after pollination
As compared to the Wild Type activities of mRNA corresponding to AT5G03220, activities are affected by the mutation in the Lec1 Mutants.

7. Who wants to Knockout AT5G03220? T-DNA Madison Screens
~612 bp
~408 bp
~500 bp
500
Knockout candidates for AT5G03220 were identified to be contained in Superpool 1 (left), DNA Pool 6 (right) of the Madison Lines.
Sequencing results were repetitively unsuccessful in giving definitive results. Expected sizes of amplified product were predicted to be between 400 and 615 bp in length from Autoradiography.

8. Using SALK Lines to Genotype
SALK line was chosen due to its position in a 5’UTR of the gene.
Plants exposed to the T-DNA were assayed for Wild Type and Mutant alleles in hopes to find an embryo lethal form of the gene.
It was determined with the first ten extracted DNA samples that their genotypes were all homozygous Wild Type.

9. Is this T-DNA Insertion Embryo Lethal?
All ten samples displayed the presence of at least one Wild Type Allele, and through T-DNA specific PCR, it was verified that all of the plants were homozygous for the Wild Type allele.
So far, results obtained suggests the high possibility that the SALK insertion may cause embryo-lethality in gene AT5G03220.
Further assays on a second set of extracted DNA samples will serve to create a more robust set of data from which to draw a definitive conclusion as will dissecting siliques to provide phenotypes to the genotypic descriptions obtained.

10. The fight ain’t over yet!
Hopefully, with the verification with the next set of DNA of embryo-lethality, the focus will shift to a more in depth study of AT5G Applications in agriculture have enormous potential in terms of improving seed viability through the research of these knockouts.