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