HC70AL Symposium

1
HC70AL Symposium

• By Bensen Fan

2
GENE 1

• a.k.a. At5G51800

3
Basic Information

• Chromosome 5
• Gene 51800
• BP 21065417-21069238 3821 bp
• Orientation 5 - 3

4
Gene Structure
At5G51800
At5G51810
At5G51795
5
3
5
5
3
3
3996 bp
3175 bp
3821 bp
1112 bp
1621 bp
EXON 1
EXON 2
UTR
UTR
EXON 3
EXON 4
5
3
126 bp
971 bp
60 bp
1439 bp
106 bp
450 bp
242 bp
347 bp
79 bp
5
Protein Encoded

• Kinase-Like Protein
• 988 Amino Acids

6
Protein Kinase Family

• Modifies other protein by adding Phosphate groups
• Results in functional change

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T-DNA Information

• Genotyped 29 Salk plants
• 18 WT Plants
• 11 Heterozygous T-DNA Plants
• 0 Homozygous T-DNA Plants
• Predicted Length
• WT 335 bp
• T-DNA 321 bp or 314 bp

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Gel Picture of Genotyping
Heterozygous T-DNA (500 600 bp)
100 bp
Plant 11
Negative
Plant 10
Plant 12
Plant 13
Plant 14
Plant 15
Positive
Plant 5
Plant 1
Plant 2
Plant 3
Plant 4
Plant 6
Plant 7
Plant 9
WT
WT
Plant 1 - 15
Expected WT 335 bp Expected T-DNA lt325 bp
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Genotyping Continued
100 bp
Negative
Plant 18
Plant 16
Plant 17
Plant 19
Plant 20
Plant 21
Plant 22
Plant 23
Plant 24
Plant 25
Plant 26
Plant 27
Plant 28
Plant 29
Plant 30
Positive
WT
Plant 16 - 30
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Genotyping Continued
100 bp
1000 bp
Heterozygous?
Plant 20
Negative
Plant 16
Plant 21
Plant 18
Plant 17
Plant 23
Plant 24
Plant 29
Plant 30
Plant 22
Positive
WT
Genotyping Clarification PCR
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T-DNA Sequencing

• T-DNA Sequencing Analysis
• 4 sequences
• Total Alignment
• Inconclusive Partial Alignment
• Conclusion Unknown Site of T-DNA Insertion
• Prediction Insertion Site does NOT lie between
  the Fw and Rv Primer Sites

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Phenotype

• No Obervable Phenotypical differences between
  Heterozygous T-DNA Plant and WT Plants
• Silique and Leaf Observation
• Silique No Observable Differences
• Leaf No Observable Differences

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Nomarksi Wild Type
Axis
Cotyledon
Fully Mature
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Nomarski Mutant Seed 1
Axis
Cotyledon
Heart Stage
15
Nomarski Mutant Seed 2
Cotyledon
Axis
Torpedo Stage
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Gene Expression RT-PCR
100 bp
Tubulin (475 bp)
Seed RT
Seed - RT
Leaf RT
Leaf - RT
Negative
Positive
Expected Band 258 bp
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Gene Chip Result

• Inconsistent in the Endosperm at the Globular
  Stage
• Absent EVERYWHERE ELSE
• Agrees with RT-PCR

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Conclusion

• Encodes for a Kinase-Like Protein
• May cause seed lethality
• Expressed in the Endosperm at the Globular Stage

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GENE 2

• a.k.a. At2G40470

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Basic Information

• Chromosome 2
• Gene 40470
• BP 16909382 - 16910730 1348 bp
• Orientation 5 - 3

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Gene Structure
At2G40470
At4G40460
At2g40475
5
3
5
5
3
3
3257 bp
1023 bp
1348 bp
1221 bp
4374 bp
UTR
EXON1
EXON 2
EXON 3
UTR
5
3
106 bp
16 bp
259 bp
402 bp
126 BP
158 bp
284 bp
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Protein Encoded

• Lateral Organ Boundaries (LOB) Domain-Containing
  Protein 15
• 223 Amino Acids

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Lateral Organ Boundaries (LOB)

• Unknown Function
• Expressed in branches, leaves and roots.
• Domain present in 42 other Arabidopsis proteins

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T-DNA Info

• Genotyped 15 SALK Plants
• 12 WT Plants
• 1 Heterozygous Plants
• 2 Homozygous Plants
• Predicted Length
• WT 930 bp long
• T-DNA 580 bp or 650 bp long

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Gel Picture of Genotyping
lt800 bp
900 1000 bp
Expecting WT 930 bp T-DNA 430 bp or 500bp
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T-DNA Sequencing

• T-DNA sequencing analysis
• Reverse Primer
• Total 657 bp long
• 514 bp lines up with WT sequence
• 143 bp dont line up with WT sequence
• Conclusion T-DNA insertion Site is 586 bp from
  the Rv Primer site
• T-DNA 736 bp long

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Phenotype

• No observable phenotypical differences between
  Homozygous T-DNA plants and WT plants
• Silique and Leaf observation
• Silique No observable differences
• Leaf No observable differences

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Microscopy Silique
T-DNA Silique
WT Silique
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Microscopy Seeds
WT Seeds
T-DNA Seeds
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Gene Expression RT-PCR
100 bp
Tubulin
Seed RT
Seed - RT
Leaf RT
Leaf - RT
Negative
Positive
Expected Size 232 bp
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Gene Chip Data

• Expressed
• High General Seed Coat in the Globular
  Stage
• Medium Root at the Vegetative Stage
• Medium Stem at the Vegetative Stage
• Low Seed at the Globular Stage
• Absence everywhere else
• Agrees with RT-PCR Result

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Promoter Cloning

• Upstream Region
• 3600 bp
• Expecting 6.2 kb bands

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First Ligation
1000 bp
6000 bp
3000 bp
1600 bp
Undigested 1
Undigested 2
Digested 2
Digested 1
Digested 3
Digested 4
Digested 5
Digested 6
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Second Inoculation
1000 bp
6000 bp
3000 bp
1600 bp
Digested 10
Digested 15
Digested 18
Digested 11
Digested 12
Digested 13
Digested 14
Digested 16
Digested 17
Digested 9
Digested 7
Digested 8
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Second Ligation
1000 bp
6000 bp
1600 bp
Digested 20
Digested 19
Digested 21
Digested 22
Digested 23
Digested 24
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Conclusion

• Encodes for LOB Domain-Containing Protein 15
• Does not cause seed lethality
• Causes no observable phenotypical variations
• Expressed in Seeds, Roots, and Stems

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Acknowledgement

• Thank you to every participants, students and
  faculties alike, of HC70AL Spring 2008
• Special Thanks to
• Anhthu
• Bekah
• Daisy
• Brandon
• Tomo