Determining Functionality of Arabidopsis Thaliana Genes in Embryo Development

1
Determining Functionality of Arabidopsis Thaliana
Genes in Embryo Development

• Ria Yagnik

2
AT2G33480
Courtesy of
Protein Info
Gene Info

• 268 amino acids
• Part of the NAC protein family
• Sub-family NAM (no apical meristem)
• Is a transcription factor

• 1196 base pairs (bp) long
• 3 exons, 2 introns
• Oriented 5 ? 3 with
• respect to chromosome

3
Primers and Band Sizes
Predicted wild-type band size (FW/RV) 1181
bp Predicted T-DNA band size (RV/LBb1) 1009 bp
LBb1
T-DNA
FW
RV
Note primers are not to scale with gene
4
Determining Genotypes

• 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

Genotyping PCR
Size 1.2 kb matches expected results
Isolated gDNA
Size 300 bp ??? try expt. again
5
Determining Genotypes

• 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

Genotyping PCR
Size 1.2 kb matches expected results
Isolated gDNA
Size 300 bp
6
Separation of Primers
FW/RV
RV/LBb1
FW/LBb1
Thus, T-DNA is oriented in the reverse direction,
contrary to what SALK thought
7
Where is gene active?
8
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.

9
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)

Predicted promoter size 2573 bp
Digested plasmid colonies
(contains excess EtBr)
10
Looking at the plant
WT
Mutant
No observable phenotypic difference
11
AT5G13180
Courtesy of
Protein Info
Gene Info

• 252 amino acids
• Part of the NAC protein family
• Sub-family NAM (no apical meristem)
• Is a transcription factor

• 1273 base pairs (bp) long
• 3 exons, 2 introns
• Oriented 5 ? 3 with
• respect to chromosome

12
Primers and Band Sizes
Predicted wild-type band size (FW/RV) 991
bp Predicted T-DNA band size (RV/LBb1) 624 bp
LBb1
T-DNA
FW
RV
Note primers are not to scale with gene
13
Determining Genotypes
Size .9 kb matches expected results
Size .6 kb matches expected results
Isolated gDNA
Genotyping PCR
Size 6 kb confirms mutant
14
Where is gene active?
15
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.

16
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)

Predicted promoter size 3016 bp
Digested plasmid colonies
17
Looking at the plant
WT
Mutant
No phenotypic difference
18
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.
19
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