Lecture 8
Genes and traits of interest
Neal Stewart
Happy Valentine’s Day!
1.
Objectives and discussion
questions
What is genomics (and “omics” in general)? What are the tools for
finding genes that might be useful in biotechnology.
2.
Know the principles of two key methods: cDNA (complementary
DNA) library production and PCR (polymerase chain reaction).
3.
What are the differences between “input” and “output” traits?
Considering the environmental and biological factors that limit
production in a farmer’s field, what are some new input traits that
might be good candidates for improvement using biotechnology?
4.
Consider the possibility that you are employed by an agricultural
biotechnology company, and they ask you to find a bacterial gene
for resistance to a specific herbicide. The herbicide has been
manufactured by the company for many years. Using a strategy
similar to that used to find glyphosate resistance, where might you
start to look for a bacterium resistant to that herbicide?
More objectives
5. Golden Rice producing provitamin A has the potential to help many
impoverished people who might benefit from eating it. Although
application of this technology is supported by many people and
organizations, there are also some who oppose the technology.
Considering their possible motivations and potential biases, discuss
some of the reasons that groups have come out in favor or in
opposition to Golden Rice.
6. What are the potential benefits of producing pharmaceutical proteins
in plants? What are some of the disadvantages or potential
dangers?
7. Animal genes can be inserted into plants and expressed. Would you
be opposed to eating foods from plants expressing proteins encoded
by animal genes? By human genes? Discuss the reasons for your
answers.
Finding genes to modify: many
strategies
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Biochemistry—understanding pathways
Functional screens
Hunting for specific genes– cDNAs
Searching databases for similar genes
Omics
How plant genome sizes compare
with other organisms
http://fig.cox.miami.edu/~cmallery/150/gene/genome2.jpg
Omics—what’s the point?
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Genomics
Transcriptomics
Proteomics
Metabolomics
Phenomics
Omics—what’s the point?
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Genomics
Transcriptomics
Proteomics
Metabolomics
Phenomics
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ID genes
Transcript abundance
Protein abundance
ID metabolites
To understand how
perturbing the system
results in a new
phenotype—whole
plant phenotyping
Omics—main approaches
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Genomics
Transcriptomics
Proteomics
Metabolomics
Phenomics
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Genome sequencing
RNA-seq
Mass spec techniques
Mass spec techniques
Suite of technologies
that can be automated
and used in the field
Creating cDNAs
complementary DNA from mRNA
Introducing PCR
Figure 7.10
http://youtube.com/watch?v=_YgXcJ4n-kQ
Test material
Sample preparation
Reference material
Source: Murali Rao
Total RNA isolation
mRNA purification
Image Collection
Sample Array Data
Progression of GM plants
• 1st Generation: Input traits (herbicide
tolerance, insect resistance, etc.)
• 2nd Generation: Output traits: (enhanced
nutrition, etc.)—also known as quality traits
• 3rd Generation: Non-traditional–
(pharmaceuticals, phytoremediation,
phytosensors, next-generation biofuels)
First generation
Input traits
Herbicide-resistant crops
Figure 8.1
Finding a glyphosate resistance gene
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Discovery of glyphosate—kills most plants
Knowledge of shikimate pathway
Targeting enyzme (gene) for resistance
Functional screen for resistance
Clone resistance gene
Overexpress gene in plants
Discussion question
Consider the possibility that you are
employed by an agricultural biotechnology
company, and they ask you to find a
bacterial gene for resistance to a specific
herbicide. The herbicide has been
manufactured by the company for many
years. Using a strategy similar to that used
to find glyphosate resistance, where might
you start to look for a bacterium resistant
to that herbicide?
Transgenic disease resistance
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Viruses (yes)
Bacteria (no)
Fungi (no)
Nematodes (no)
Photo by Dennis Gonsalves.
Stewart, 2004. Genetically Modified Planet 2004
Figure 8.5
Biotechnologist of the day
Dennis Gonsalves
• From Hawaii
• At USDA-ARS in
Hilo, Hawaii
• Humboldt Prize
winner 2002
• Virus resistant
papaya credited
for saving papaya
industry in Hawaii
Figure 8.4
Insect resistance
Controlling Colorado potato beetle
is not easy
Bt corn
Bt cotton
Bacillus thuringiensis
Stewart, 2004. Genetically Modified Planet 2004
Bt Cry structure
III
I
II
Stewart, 2004. Genetically Modified Planet 2004
Figure 8.3
Bt toxin
Insect midgut cells that have bound Bt toxin.
Same gut cells a few hours later– note the damage and leakage.
Stewart, 2004. Genetically Modified Planet 2004
Bt
Insect midgut cells that have bound Bt toxin.
Mutated receptors cannot bind Bt toxin.
Stewart, 2004. Genetically Modified Planet 2004
Receptors are not present– cells cannot bind Bt
Different Bt Crys
• Cry 1s—kills
caterpillars
(lepidoptera)
• Cry 2s—kills
caterpillars
(lepidoptera)
• Cry 3s—kills beetles
(coleoptera)
Canola plant expresses a Bt cry1Ac gene
Discussion question
Other than the products discussed in this
chapter, what other sorts of genes or
strategies might be useful in engineering
transgenic plants resistant to insects or
pathogens?
Second generation
Output traits=quality traits
Improved nutrition, better foods
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Golden rice
Modified oils from oilseeds
Vitamin E enhancements
Arctic apple—non-browning
Innate potato—non-browning and reduced
acrylamide upon browning
Golden rice: producing provitamin A
Biotechnologist of
the day:
Ingo Potrykus
Figure 8.6
Third generation
Non-traditional products
Examples
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Pharmaceuticals
Oral vaccines
Phytoremediation
Phytosensors
Biofuels
Plant-made pharmaceuticals
aka
Molecular pharming
Duckweed
Genetic
Engineering
Grow in lab
Or field
Protein
Purification
Oral vaccine– eat the fruit, or purify the
vaccine pill or injection
Corn
Fraunhofer USA: one plantbased platform to produce
pharmaceutical proteins:
vaccines
http://www.youtube.com/watch?v=
oCGFW1WOFTY
What are the potential benefits of
producing pharmaceutical
proteins in plants? What are some
of the disadvantages or potential
dangers?
Phytorediation example
How to remediate mercury in soil
www.uga.genetics.edu/rmblab
So, transgenic plants could be
used in a lot of applications…
Are there any we should avoid?
PCR videos!
How PCR works:
http://youtube.com/watch?v=_YgXcJ4n-kQ
PCR song!
http://youtube.com/watch?v=x5yPkxCLads&feature=related