Determining Functionality of Arabidopsis Thaliana Genes in Embryo Development
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Transcript of Determining Functionality of Arabidopsis Thaliana Genes in Embryo Development
Determining Functionality of Arabidopsis Thaliana
Genes in Embryo Development
Ria Yagnik
AT2G33480Gene Info
• 1196 base pairs (bp) long• 3 exons, 2 introns• Oriented 5’ 3’ with respect to chromosome
• 268 amino acids• Part of the NAC protein family• Sub-family = NAM (no apical meristem)• Is a transcription factor
Protein Info
1 68 252 330 583 673 1042 1196
intron exon UTR
5’ Chromosome/gene direction 3’
T-DNA
151
Courtesy of:
Primers and Band Sizes
FW »» «« RV
LBb1 »»
Note: primers are not to scale with gene
Predicted wild-type band size (FW/RV) = 1181 bp
Predicted T-DNA band size (RV/LBb1) = 1009 bp
T-DNA
Determining Genotypes
Isolated gDNA
Genotyping PCR
• DNA was isolated from plant leaves and fractionated on a gel to confirm the presence of both DNA and RNA• A 0.2 concentration of that DNA stock was then amplified using PCR, gene specific primers and Hox7D primers as a control
Size ~ 1.2 kbmatches expected results
Size ~ 300 bp ??? try expt. again
Isolated gDNA
Genotyping PCR
• DNA was isolated from plant leaves and fractionated on a gel to confirm the presence of both DNA and RNA• A 0.2 concentration of that DNA stock was then amplified using PCR, gene specific primers and Hox7D primers as a control
Size ~ 1.2 kbmatches expected results
Size ~ 300 bp
Determining Genotypes
Separation of Primers
FW/RV RV/LBb1 FW/LBb1
Thus, T-DNA is oriented in the reverse direction, contrary to what SALK thought
Where is gene active?
Where is gene active?
• As seen by the GeneChip data, mRNA of my gene is present in smaller amounts in the leaf than the silique• However, the leaf amplification is much greater than the silique, suggesting more leaf RNA was present to become a greater quantity of cDNA• This could be due to various factors, such as age of the leaf/silique, quality of RNA extracted, etc.
Cloning the Promoter Region
Band locations• 3.5 kb (TOPO vector)• 2.6 kb (promoter region)• Various other band fragments (different stages of partial digestion)
Digested plasmid colonies
(contains excess EtBr)
Predicted promoter size: 2573 bp
Looking at the plant
No observable phenotypic difference
WT
Mutant
AT5G13180Gene Info
• 1273 base pairs (bp) long• 3 exons, 2 introns• Oriented 5’ 3’ with respect to chromosome
• 252 amino acids• Part of the NAC protein family• Sub-family = NAM (no apical meristem)• Is a transcription factor
Protein Info
1 67 248 343 590 673 1003 1273
intron exon UTR
5’ Chromosome/gene direction 3’
T-DNA
53
Courtesy of:
Primers and Band Sizes
FW »» «« RV
LBb1 »»
Note: primers are not to scale with gene
Predicted wild-type band size (FW/RV) = 991 bp
Predicted T-DNA band size (RV/LBb1) = 624 bp
T-DNA
Determining Genotypes
Isolated gDNA Genotyping PCR
Size ~ .9 kbmatches expected results
Size ~ .6 kb matches expected results
Size ~ 6 kb
confirms mutant
Where is gene active?
Where is gene active?
• In the GeneChip data, we see that the mRNA of my gene is present in smaller amounts in the leaf and silique• However, the leaf amplification (left) is much greater than the silique (lane 3), suggesting more leaf RNA was present to become a greater quantity of cDNA• This could be due to various factors, such as age of the leaf/silique, quality of RNA extracted, etc.
Cloning the Promoter Region
Band locations• 3.5 kb (TOPO vector)• 3.0 kb (promoter region)• Various other band fragments (different stages of partial digestion)
Digested plasmid colonies
Predicted promoter size: 3016 bp
Looking at the plant
No phenotypic difference
WT
Mutant
The Big Question
Is my gene critical for embryo development?
Answer: From current research, AT2G33480 and AT5G13180 of Arabidopsis thaliana do not appear to be critical in the formation of embryos or seeds.
However, further research must be done (for example, using multiple knockouts to account for redundancy) before their application during
development can be fully determined.
Acknowledgements
I would like to acknowledge the many many individuals who provided countless hours of their own time helping me with
this project.
Tomokazu Kawashima
Brittan Starr Scales
Mike Gaviño as well as our amazing HC70AL
Dr. Anhthu Bui class: Emily, Yosuke, Rena, Jon
Dr. Xingjun Wang Eric, Combiz, Tim, Joanna,
and of course Yuya, Mike and Garen
Dr. Bob Goldberg