Molecular mapping of an ATP insensitive Arabidopsis thaliana mutant

Blake Stephens

• Missouri Academy of

Science Mathematics

and Computing

• Northwest Missouri State

University

• Missouri Academy

Research Summer

(MARS)

• Deanna Lankford

Graduate with:

1. High School Diploma from

Plattsburg High School

2. Associates Degree of

Science from Northwest

Missouri State University

• ATP plays an important role as an extracellular signaling molecule.

• Purinergicreceptors are responsible for extracellular ATP responses in mammals, but these same receptors seem to be lacking from plant genome.

It is apparent that

similar responses occur

in plants.

Jaffe (1973) Plant Physiol 51:17-18

ATP stimulates closure of the Venus flytrap

To contribute to the discovery and categorization of

the plant ATP response system.

• ATP-insensitive

Mutant

• Wild-Type

11-4

11-4

11-4WT

• Using a forward

genetic

approach we

are identifying

the

chromosome on

which the gene

is present.

Seeds were harvested

from decedents of

different crosses

between the WT and

11-4 organisms. The

seeds were cleaned

and then sowed in a

MS Agar Media.

Earlier we

mentioned plant

responses to ATP

similar to those

found in

mammals.

One such

response is an

increase in

cytosolic calcium

concentrations

when ATP is

released into the

plant’s

environment.

Using a computer program I wrote, the data was

normalized and converted automatically into an Excel-

readable file, saving much time on what was previously a

largely manual process.

Here are some

examples of what the

normalized data

looks like when

graphed.

Wild Type

Mutant

Non-Transgenic

Concentration (uM) Concentration (uM) Concentration (uM)

Plate 1 NoR HiR LoR NS Plate 7 NoR HiR LoR NS Plate 13 NoR HiR LoR no sig

1 = WT 3 8 0 1 = 6-2 0 5 3 4 1 = 4-1-1 0 4 8 0

2 = 11-4mut 10 2 0 2 = 6-3 1 0 1 10 2 = 4-1-2 0 4 8 0

3 = 1-1 7 3 2 3 = 6-4 0 3 6 3 3 = 4-1-3 8 1 3 0

4 = 1-2 2 5 5 4 = 7-1 2 3 3 4 4 = 4-1-4 2 5 5 0

5 = 1-3 4 3 5 5 = 7-2 4 2 3 3 5 = 4-2-1 8 0 4 0

6 = 1-4 5 2 5 6 = 8-1 0 1 7 4 6 = 4-2-2 1 5 6 0

7 = 1-5 4 5 3 7 = 8-2 2 4 4 2 7 = 4-2-3 8 1 3 0

8 = 1-6 7 4 1 8 = 8-3 0 3 5 4 8 = 4-2-4 3 1 8 0

Plate 2 NoR HiR LoR NS Plate 8 NoR HiR LoR NS Plate 14 NoR HiR LoR NS

1 = 8-4 3 2 3 4 1 = 4-3-1 4 3 3 2

2 = 9-2 0 0 2 10 2 = 2 1 6 5 0

3 = 9-4 2 5 3 2 3 = 4 8 3 1 0

4 = WQ 0 12 0 0 4 = 5-1-1 1 8 3 0

5 = 2 3 5 4 0

6 = 3 0 5 7 0

7 = 4 0 3 9 0

8 = 5-2-1 0 3 9 0


1 = W 0 0 0 12 1 = 5-2-2

Key 2 = 11-4 0 0 0 12 2 = 3

Homo Mut 3 = 2-1-1 3 = 4

Homo Wild 4 = 2-1-2

Poss. Hetero 5 = 2-1-3

6 = 2-1-4

7 = 2-2-1

Plate 4 NoR HiR LoR NS 8 = 2-2-2

1.1 = WQ 0 4 2 0 Plate 10 NoR HiR LoR NS Plate 16 NoR HiR LoR NS

1.2 = 11-4 3 2 1 0 1 = 2-2-3 0 7 5 0 1 = 3-2 8 0 1 3

2 = 2-1 2 3 5 2 2 = 2-2-4 0 12 0 0 2 = 3-7 0 0 0 12

3 = 2-2 1 7 1 3 3 = 2-3-1 0 12 0 0 3 = 4-2 2 0 3 7

4 = 2-3 1 7 2 2 4 = 2-3-2 0 12 0 0 4 = 12-2 2 3 1 6

5 = 2-4 2 2 6 2 5 = 2-3-3 0 9 3 0 5 = 12-1 9 1 2 0

6 = 2-5 1 5 3 3 6 = 2-3-4 0 11 1 0 6 = 12-3 2 2 2 6

7 = 2-6 1 7 2 2 7 = 2-4-1 0 6 6 0 7 = 12-4 9 0 2 1

8 = 2-7 2 3 4 3 8 = 2-4-2 0 5 7 0 8 = 13-1 8 1 1 2


1 = 2-9 0 0 0 12 1 = 2-4-3 3 2 7 0 1 = 14-1 7 0 2 3

2 = 2-8 5 0 0 7 2 = 2-4-4 9 3 0 0 2 = 2 6 0 5 1

3 = 2-10 3 2 1 6 3 = 3-1-1 10 1 1 0 3 = 15-1 0 0 6 6

4 = 2-11 3 2 0 7 4 = 2-5-1 9 1 2 0 4 = 2 9 1 0 2

5 = 2-12 0 11 1 0 5 = 3-1-2 12 0 0 0 5 = 3 3 2 4 3

6 = 2-13 8 0 2 2 6 = 3-1-3 6 0 6 0 6 = 4 6 2 1 3

7 = 2-14 1 9 3 0 7 = 3-1-4 7 0 5 0 7 = 16-1 6 0 1 5

8 = 2-15 5 2 1 4 8 = 3-2-1 10 0 2 0 8 = 2 5 1 0 6


1 = 2-16 3 1 6 2 1 = 3-2-2 11 0 0 1 1 = 16-6 2 3 2 5

2 = 2-17 0 6 2 4 2 = 3-2-3 8 1 3 0 2 = WQ 4 4 2 2

3 = 2-18 2 4 4 2 3 = 3-3-1 2 4 2 4

4 = 2-19 2 7 3 0 4 = 3-3-2 0 3 1 8

5 = 2-20 0 2 10 0 5 = 6-1 11 0 1 0

6 = 2-21 1 0 2 9 6 = 6-2 3 0 1 8

7 = 2-22 2 5 1 4 7 = 6-4 9 1 2 0

8 = 2-23 2 2 4 4 8 = 3-2-4 5 5 2 0

This is a

chart

descriptiv

e of the

phenotypi

c natures

of each

genetic

line.

• The specimens that were

most likely homozygous-

mutants were selected

for genetic testing.

• PCR was used to amplify

specific areas of the

gene.

• A four-percent gel-

agarose was used in a

gel electrophoresis

process to separate and

categorize each

specimen’s genotype.

Col Het Ler Col Het Col Het Het Ler Het Col Het

CONTROL

Gel from nga172

These charts represent the gene and how many of our

specimens shared the genotype of the 11-4 organism.CHROMOSOME NAME TEMP #Col #H+L #Het #Lan RECOMB CHI-SQUARE RATING Temp

nga63 55 0 #DIV/0! 0.007090101 48

ciw12 48 1 8 5 3 0.611111111 0.379589586 Great 52

SO392 55 0 #DIV/0! 0.007090101 55

CHROM 1 ciw1 48 0 #DIV/0! 0.007090101 Ind.

nga280 55 3 6 2 4 0.555555556 0.495056708 Great Rating

NF5|14 52 1 8 6 2 0.555555556 0.379589586 Great Ind. (Indescernable)

ATPase 55 2 7 1 6 0.722222222 0.848694963 Great Bad

nga1145 55 3 0 0 0 0 0.006169899 Bad Poor

CHROM 2 ciw3 48 0 0 0 0 #DIV/0! 0.002054719 Poor Ind. Good

nga1126 55 2 7 2 5 0.666666667 0.848694963 Poor Great

nga168 55 2 0 0 0 0 0.006981285 Bad

ng172 52 2 7 5 2 0.5 0.848694963 Great Gene?

nga126 52 0 0 0 0 #DIV/0! 0.002054719 Ind. LOW

CHROM 3 nga162 52 3 6 5 1 0.388888889 0.495056708 Good

ciw11 48 0 0 0 0 #DIV/0! 0.002054719 Ind.

ciw4 52 7 2 0 2 0.222222222 0.000200168 Good MED

nga6 52 3 4 0 4 0.571428571 0.191388523 Poor

ciw5 48 1 8 6 2 0.555555556 0.379589586 Great

nga8 52 6 2 2 0 0.125 0.001522054 Great HIGH

CHROM 4 ciw6 52 3 6 1 5 0.611111111 0.495056708 Great IND.

ciw7 48 0 #DIV/0! 0.007090101

nga1139 52 2 7 4 3 0.555555556 0.848694963 Good

nga1107 48 5 4 1 3 0.388888889 0.028163881 Good

nga225 52 8 1 1 0 0.055555556 7.41771E-06 Bad

nga249 52 4 5 3 2 0.388888889 0.151271016 Good

nga151 52 4 5 3 2 0.388888889 0.151271016 Great

CHROM 5 nga139 55 0 #DIV/0! 0.007090101

PHYC3 52 2 1 1 0 0.166666667 0.019956349 Bad

ciw9 52 3 5 1 4 0.5625 0.33015855 Great

ciw10 48 0 #DIV/0! 0.007090101

1-4 2-13 2-20 3-2 12-4 13-1 14-1 15-2 12-1

nga63

ciw12 c l h h h h l l h

SO392

ciw1

nga280 c l h l h c l l c

NF5|14 c h h l h h h l h

ATPase c h c l l l l l l

nga1145 c l l l l c l c

ciw3

nga1126 c h l h l l l l c

nga168 c l l l l l c l h

ng172 c h c h h l h l h

nga126

nga162 c c c h h l h h h

ciw11

ciw4 c c c l c c c c l

nga6 c c l l c? c? l c l

ciw5 c h h l h h h l h

nga8 c c c c c c c c

ciw6 c l l h l l c l c

ciw7

nga1139

nga1107 c c l h c l c c l

nga225 c c c l c c c c c

nga249 c c l l h c h c h

nga151 c h l l h c h c c

nga139

PHYC3

ciw9 c c l? h c l l l l

ciw10

After analyzation of the data, it was determined that the gene of interest was located close to the nga8 region on the fourth chromosome.

• Continue mapping with more specimens to further clarify

the data and ensure its accuracy.

• Continue mapping with different markers to get high-

resolution mapping results.

• Engineer a program to fully automate the normalization

and graphing process used in this experiment.

• Perform whole genome sequencing using BC1F3 back-

cross line to identify mutation point.

For your time today, as well as

for these interesting and

educational past two months. I

have gained useful lab

experience, and been given a

unique insight into the world of

research.

To Kiwamu

Tanaka,

Deanna

Lankford, Gary

Stacey, and the

members of the

Stacey Lab.

Technology

Molecular mapping of an ATP insensitive Arabidopsis thaliana mutant