HC70ALFinal Presentation

Chris McQuilkinJune 4th, 2009

Gene One: AT4G36540

• A transcription factor involved in DNA binding

• A bHLH Gene – “basic Helix-Loop-Helix” refers to the

structure of the protein– bHLH genes are found in Eukaryotes and

are highly conserved across species

AT4G36540What is the Structure of the Gene?

116 288 85 180 92 207 91 134 91 93 331

Exon 1 Exon 5 Exon 4Exon 3 Exon 2

Translational Start Codon

Translational Stop Codon

Intron 1 Intron 2 Intron 3 Intron 4

Total length= 1,714 base pairs

T-DNA Insertion Site

Forward Primer

Reverse Primer

LB

Where is the Gene Active?

CM Arabidopsis RT-PCR Gene: AT4G36540 (Leaf and Silique cDNA)

ScD

NA

GD

NALcD

NA

10

0 bp

•Why is there no band in the positive control?

RT-PCR Forward Primer

Forward Primer

Intron

Where is the Gene Active?mRNA accumulation

0

500

1000

1500

2000

2500

Chala

zal S

eed

Coat/G

lobu

lar S

tage

Chala

zal S

eed

Coat/P

regl

obul

ar S

tage

Flor

al B

ud/R

epro

duct

ive

Leaf

/Veg

etat

ive

Ovule

/Rep

rodu

ctive

Seed

/24-

Hr Pos

t-Fer

tiliza

tion

Seed

ling/

3 Day

s Afte

r Im

bibi

tion

(3DAI)

Stem

/Veg

etat

ive

Seed

/Pre

glob

ular

Sta

ge

Plant region/ stage of development

mR

NA

Acc

um

ula

tio

n

Series1

Where is the Gene Active?Promoter Cloning

Strategy of Promoter ActivityStrategy of Promoter ActivityAnalysisAnalysis

Arabidopsis Genomic DNA

•PCR amplification of upstream region

•With Gene-specific Primers

•And High Fidelity DNA Polymerase

PCR Product pENTR/D-TOPO vector

Ligation: Population of Recombinant Plasmid(vector+PCR product) and NON-recombinant plasmid(vectory only)

Transformation of competent E.coli cells

Screening for E.coli cells harboring recombinantplasmid

Confirmed Recombinant plasmid DNA: Verifying the authenticity ofrecombinant plasmid DNA by Restriction Enzyme Digestion

DNA sequences: verification of the clonedPromoter Region by Sequencing Analysis.

Sequence Analysis and confirmed identity of thecloned upstream region

Recombinant Plasmid DNA + Beta-Gluronidase (GUS) gene carrying T-DNAVector

Krista Templeton and Auni Hovanesian, HC70AL 2008

Promoter Cloning*Two fragments were amplified by PCR

PCR of AT4G3540 Promoter Region

1 hr 120 volts

1 k

b La

dd

er

iPro

of

Poly

mera

se

Positiv

e C

on

trol

~1.7 kb= Expected Size of PCR-amplified promoter~0.8 kb= Unexpected PCR product

Expected Plasmid alone= 2.5 kb

Expected Plasmid + Promoter= 4.2kb

Observed= 2.5kb and 3.5 kb

Promoter Cloning

AscI-Digested pENTR Plasmid DNA From Six E. coli Colonies

Gene one: Genotyping

1 hr, 120 volts

Wild-type Control

04/16/2009

1 hr, 120 volts

Gene one: Genotyping

Hom

ozy

go

us

Hem

izygous

Hem

izygou

s Hem

izygous

What is the expected size of the T-DNA band?

887 Bases

What is the expected size of the Wild-Type Band?

~250 bases

~250 base pairs

~900 base pairs

~1000 base pairsWhy are there two bands in the Lanes 1 and 3?

Concatamers

119 288 85 180 92 207 91 134 91 93 334

Exon 1 Exon 5 Exon 4Exon 3 Exon 2

Translational Start Codon

Translational Start Codon

Intron 1 Intron 2 Intron 3 Intron 4

116 288 85 180 92 207 91 134 91 93 331

Exon 1 Exon 5 Exon 4Exon 3 Exon 2

Translational Start Codon

Translational Start Codon

Intron 1 Intron 2 Intron 3 Intron 4

T-DNA Insertion Site

Forward Primer

Reverse Primer

LB LB

Nomarski Observation

Mutant Embryo and wild-type embryo show no phenotypic differences

Nomarski Observation

Mutant seed coat and wild- type seed coat show no phenotypic differences

Results

• Three hemizygous and one homozygous T-DNA plants were identified– Two bands were observed in each of the

lanes containing DNA with a T-DNA insert

• Gene AT4G36540 mRNA accumulation was observed in both the leaf and silique of Arabidopsis

• The promoter did not insert into any of the E. coli plasmids that were screened.– Four of the colonies contained an unidentified

insert about 1 kb long

• No phenotypic changes were observed in either the hemizygous or homozygous T-DNA Arabidopsis plants using Nomarski Observation

Results

Conclusion

• AT4G36540 is not lethal, or there may be another DNA sequence that codes for the same protein

• Although no phenotypic differences were observed in the seed coat or embryo using Nomarski, there could be differences that are more subtle or appear at different stages of development

What next?

• Look further for phenotypic changes in Arabidopsis mutants—examine different tissues and more stages of development

• Sequence the unidentified DNA fragment taken up by the E. coli C600 plasmids

• Repeat PCR of the promoter– Excise the band containing recombined plasmid DNA, and

repeat transformation– If a promoter is identified, use GUS to study where the gene is

expressed• Grow more plants from the identified mutant lines• Repeat sequencing reaction of region between LB and

T-DNA primers to confirm location of the T-DNA insert

Gene Two: AT1G75240

• A homeobox gene

• Homeobox genes regulate development and cell differentiation– A homeobox is a sequence

• They are found in animals, plants, and fungi

AT1G75240What Is The Structure of the Gene?

330 77 97 927 254

Translational Start Codon

Translational Stop Codon

Forward Primer Reverse Primer

Total Length: 1,685 Base Pairs

Predicted T-DNA Insert

LB

Where is the Gene Active?

1 hr 120 volts

AT1G75240 mRNA in Arabidopsis Leaf and Silique

Leaf c

DN

A

Siliq

ue c

DN

A

Gene is active in the silique but not the leaf

Tubulin mRNA AT1G7524

0 mRNA

Where is the Gene Active?

mRNA Accumulation of AT1G75240

0500

100015002000250030003500

Em

bryo

Pro

per/

Hea

rt S

tage

Em

bryo

Pro

per/

Mat

ure

Ovu

le/R

epro

duct

ive

See

d/C

otyl

edon

Sta

ge (

7-8D

AP

)

See

d/M

atur

e G

reen

Sta

ge

See

d/P

ost-

Mat

ure

Gre

en S

tage

(18

-

Developmental Stages

Ave

rag

e m

RN

A A

ccu

mu

lati

on

Series1

Genotyping

Arab

ido

psis 1

Arab

ido

psis 8

Arab

ido

psis 2

Arab

ido

psis 7

Arab

ido

psis 6

Arab

ido

psis 5

Arab

ido

psis 4

Arab

ido

psis 3

Arab

ido

psis 9

Arab

ido

psis 10

Arab

ido

psis 11

Arab

ido

psis 12

WT

Co

ntro

l

100 bp

100 bp

LB

b1 C

on

trol

Results

• All the plants screened were wild-type

Conclusion

• The chance of getting all wild-type plants is extremely low, so it is possible that the Salk Institute sent the wrong kind of plant

Thank you!

• Anhthu Bui• Brandon Chen• Bob Goldberg • Daisy Robinton• Ingrid Nelson• Kristin Gill• Min Chen

SRB Contig Sequencing

Contig Length: 50.7 KB

Approach

• Contig entered into three online databases that look for predicted genes: FGENESH, GENSCAN, and GeneMark

396-507 999-10921324-1428

-2086 2126-2351 2990

1901-

281- 427 459- 572

1 2,000

2,001 4,000

628- 729 759-846 999-1097 1134-1642 1843-

2894 3046

-2550

240 396 - 507 1134 - 1442 1901 -

-2550 3272

Approach

• Predicted Genes were entered into a BLAST search to see if the predicted gene matched Expressed Sequence Tags (ESTs) found in other species

• Whole Contig was searched for DNA repeats

Approach

Results: GENSCANPredicted Gene (BLASTN)

Accession Number E-value Description

1 BP033723.1 1e-110 Lotus Japonicus

2 FN014331.1 1e-52 Petunia axillaris subsp. axillaris pool of root and petal tissue

3 GO259467 3e-26 Tissue from peanut Arachis hypogaea

4 EX522455.1 0.0 Trichome from stem of Medicago sativa

5 FK023593.1 8e-77 cDNA from Glycine max (soy bean)

Results: GENSCANPredicted Protein (tBLASTn) Accession Number E-value Description

1 Y18169 5e-70 Pisum sativum mitochondrial ccb248 gene and partial rps7 gene.

2 FM179380 2e-10 Vitis vinifera complete mitochondrial genome, cultivar Pinot noir clone ENTAV115

3 L40816 3e-35

4 DQ008791 5e-60 Thottea tomentosa large subunit ribosomal RNA gene, partial sequence; mitochondrial

5 AC144406 4e-14 Medicago truncatula clone mth2-5h18, complete sequence.

Predicted Protein (BLASTp)

4 YP_173352 2e-41 hypothetical protein NitaMp002 [Nicotiana tabacum].

5 No significant match

Results: FGENESHPredicted Gene (BLASTn)

Accession Number

E-value Description

1 EX527626 0.0 MTGland_A031_2007-05-22/MTGlandA031_B09_039_1 Medicago truncatula A17 glandular trichome Medicago truncatula cDNA, mRNA sequence

2 CD721224 2e-25 Chardonnay Vitis vinifera

3 GD544655 1e-40 Scarlet Runner Bean globular-stage suspensor region

4

5 FN014331 9e-88 Pool of root and petal tissue of Petunia axillaris

6 FF554629.1 9e-143 Vigna Unguiculata

Predicted Protein (tBLASTn)

1 Y18169 18e-118 Pisum sativum mitochondrial ccb248 gene and partial rps7 gene.

6 AC144406 8e-18 Medicago truncatula clone mth2-5h18, complete sequence

Predicted Protein (BLASTp)

1 CAB43024 2e-118 cytochrome c biogenesis protein [Pisum sativum]

Results: GeneMarkPredicted Protein (tBLASTn)

Accession Number (of Lowest E-Value)

E-value Description

1 AC192958 1e-13 Medicago truncatula chromosome 2 BAC clone mte1-45m19, complete sequence

2 AC147537 1e-23 Medicago truncatula clone mth2-133k2, complete sequence.

3 EX527626 3e-113 Glandular trichome Medicago truncatula

13 BA000042 1e-18 Nicotiana tabacum mitochondrial DNA, complete genomeLength=430597

15 L40816 5e-25 Glycine max mitochondrion polymorphic marker DNA sequence.

16 L40816 7e-46 Glycine max mitochondrion polymorphic marker DNA sequence.

17 AC145156 1e-50 Medicago truncatula clone mth2-7h6, complete sequence.

18 DQ647831 1e-108 Chlorokybus atmophyticus large subunit ribosomal RNA gene, partial

sequence; mitochondrial.

Results: Gene Mark19 BA000042 1e-41 Nicotiana tabacum

mitochondrial DNA, complete genome.

20 AP004975 1e-26 Lotus japonicus genomic DNA, chromosome 5, clone: LjT21J12, TM0158, complete sequence.

21 AP004975 7e-17 Lotus japonicus genomic DNA, chromosome 5, clone: LjT21J12, TM0158, complete sequence.

23 XM_002336124 2e-12 Populus trichocarpa predicted protein, mRNA.

32 FM179380 5e-29 Vitis vinifera complete mitochondrial genome, cultivar Pinot noir clone ENTAV115.

33 AC144406 7e-29 Medicago truncatula clone mth2-5h18, complete sequence.