Characterization of Gene Expression in
Transgenic Plants with Modified Floral Gene
Expression
Samantha Colby
Mentors: Dr. Amy Klocko and Dr. Steve Strauss
Oregon State University Undergraduate Summer Program
Why Poplar and Sweetgum?
• Poplar trees
o Wood
o Paper
o Biofuel source
• Sweetgum
o Timber
o Southeast US
o Ornamental
o Northwest US
Transgenic Plants
• Genetically modified through the use of
recombinant DNA techniques
• Insertion of advantageous genes or
modification of existing genes
• Increases diversity of plant characteristics
available to plant breeders
Genetic Engineering Defined
Traditional
Plant Breeding
x
Variety B
Variety A
Genetic
Engineering
x
Asexual
modification or
insertion from any
gene source
Containment
• Issue: the dispersal of transgenic material in
the environment
• Option: produce trees that cannot produce
viable pollen or seed
• Question: is developing an efficient, consistent
method to create sterile trees possible?
Overview
• Sweetgum
– Development of basic methods
• Poplar
– Analysis of gene expression
RNA Interference (RNAi)
• New technology
– Nobel Prize in 2006
• Turns off specific genes
– Allows one to target the genes that responsible for
forming flowers
• RNAi breaks down the molecules that make
flower proteins
General Procedure
• RNA extraction
o Qiagen RNeasy kit
• Complementary DNA (cDNA) synthesis
o Reverse transcription
• Polymerase chain reaction (PCR) using gene-specific
primers
• Gel electrophoresis to determine expression of
inserted genes
Primers: Control
Controls
Non-transgenic 2
Transgenic 1
Transgenic 2
Non-transgenic 1
Ladder
Controls
Non-transgenic 2
Transgenic 1
Transgenic 2
Non-transgenic 1
Ladder
Gel electrophoresis
Primers: Flowering (FT)
New Techniques
•
•
•
•
•
gDNA Extraction
RNA Extraction
Spectrophotometry
PCR with Gene-Specific Primers
Gel Electrophoresis
Sweetgum Project Overview
• Identify trees where RNAi is effective
– RNAi is used to turn off the flowering gene AGAMOUS
– Analyze leaf tissues to determine gene expression
Sweetgum RNA Extraction
• Attempted to extract RNA using the BioRad
sample kit
– No bands present for gel electrophoresis
– Minimal spectrophotometry nucleic acid levels
• Other RNA Extraction Methods
– RNeasy kit
– Zymogen kit
– CTAB method (standard lab protocol)
• All the above methods gave a low RNA yield
– Modified CTAB method
• Successful!
Sweetgum Primer Testing
• Tested primers for sweetgum gDNA samples
– Control and transgenic samples
– Primers specific to control and sterility genes
Ladder
Ctrl
Transgenic 1
Transgenic 2
Water
Ctrl
Transgenic 1
Transgenic 2
Water
Ctrl
Transgenic 1
Transgenic 2
Water
Ctrl
Transgenic 1
Transgenic 2
Water
Gel Electrophoresis Results
Primer 1
Primer 2
Control
Primer
Primer 3
Next Steps
• We now have an effective RNA extraction
method, as well as primers
• The sweetgum samples have been collected
from the field for further testing
Poplar Project Overview
• The lab uses RNAi to turn off 2 genes
– LEAFY and AGAMOUS
– Both genes contribute to flower formation
• The gene HSP:FT is added to help us study the
effects of RNAi
o The Heat Shock Promotor (HSP) gene allows us to turn
on FT by turning up the heat
o FLOWERING LOCUS T (FT) causes plants to flower early
o Flowering would take years without HSP:FT
HSP:FT Poplar Trees during
Heat Induction
Poplar Project Overview
• Determine the levels of FLOWERING LOCUS T
(FT) gene required to induce flowering
Poplar Project Overview
• Determine the levels of FLOWERING LOCUS T
(FT) gene required to induce flowering
Female Flower
Male Flower
Male and Female Flowering and Non-flowering Poplars
Male
Flowering
Nonflowering
Female
Flowering
Nonflowering
Male and Female Flowering and Non-flowering Poplars
Male
Flowering
Nonflowering
Female
Flowering
Nonflowering
FT Threshold Study
• Not all plants with HSP:FT flower
– Need plants that reliably make flowers so we will
know when RNAi works
• Goal: determine if there is a threshold of FT
needed for flowering
• 3 categories of sample
HSP:FT Insertion
Poplar Non-HSP:FT Control
Controls
Day 10
Day 6
Day 3
Day 0
Ladder
Over 10 Days of Heat Induction
Control primers used
Control bands
present
Poplar Flowering Group
Control Primers
Controls
Day 0
Day 3
Day 6
Day 10
Controls
Day 0
Day 3
Day 6
Day 10
Ladder
HSP:FT Insertion
Over 10 Days of Heat Induction
Flowering
Primers
Faint band for
Day 10 sample
(Flowering primer)
Poplar Non-Flowering Group
Control Primers
Flowering Primers
Controls
Sample 6
Sample 5
Sample 4
Sample 3
Sample 2
Sample 1
Controls
Sample 6
Sample 5
Sample 4
Sample 3
Sample 2
Ladder
Sample 1
HSP:FT Insertion
Samples Taken After 10 Days of Heat Induction
Non-HSP:FT Control and Flowering Group Subsets
Ladder
Ctrl, Day 0
Ctrl, Day 3
Ctrl, Day 6
Ctrl, Day 10
Control
F, Day 0
F, Day 3
F, Day 6
F, Day 10
Control
Positive FT Ctrl
Control
Positive Control Added
Control
Primers
Flowering
Primers
Faint band for
Day 10 sample
Conclusion to Poplar Project
• FT expression present in flowering poplars
only
– Correlation between presence of FT and flowering
• Next Steps
– There are many more samples to be tested for FT
expression
– If band is present for flowering primers, we can
quantify the band intensity relative to the control
bands
Relative FT Expression
Relative FT Expression (%)
16
14
12
10
8
6
4
2
0
Day 0
Day 3
Day 6
Samples
Day 10
Control
Acknowledgements
Dr. Amy Klocko
Dr. Steve Strauss
SURE Science Scholarship
Members of Strauss Lab
Cathleen Ma, Kori Ault, Elli Esfandiari, Sarah Robertson, Kelly Vining, Estefania Elorriaga