Development of super high oleic oil in safflower O6 Craig Wood SHO safflower.pdf · Molecular basis...
Transcript of Development of super high oleic oil in safflower O6 Craig Wood SHO safflower.pdf · Molecular basis...
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Craig Wood | CSIRO Plant Industry
Development of super high oleic oil in safflower
ISPL2012 SEVILLE
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Overview What is super high oleic (SHO) oil?
why is SHO a possible industrial oil
Molecular basis of high oleic (HO) safflower
a natural mutation
Molecular basis of further increases in oleic content
use of hairpin RNAi and genetic engineering
Can safflower produce SHO?
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What is super oleic oil?
• near pure oleic acid • ~95% oleic acid as a target
• no polyunsaturates • PUFA are reactive • Reduce chemical synthesis efficiencies
• reduced saturates • Increased flux towards 18:1
Triolein
Triolein
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SHO is an industrial oil
SHO can be used either:
• Directly →
• as a lubricant and heat-transfer fluids
• Biodegradable
• Stable and liquid at ambient temperatures
• Indirectly →
• processed into azelaic acid (a C9 dicarboxylic acid)
• ‘crack’ 18:1 into two C9 molecules
• Extra double bonds ruin this reaction
• Cosmetics
• Specialized plastics
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Why attempt SHO in safflower in Australia?
• Safflower has a simple oil profile
• 18:1, 18:2, 16:0, 18:0
• Non-photosynthetic seed
• Closed flowerhead structure
• Preferred structure
• Not widely grown in Australia
• Not a food crop
• GM segregation is well-established in Australia
• GM-cotton and GM-canola as precedents
• SHO is an easier GM trait for regulatory approval
• SHO is a ‘normal’ oil (18:1) but at very high purity
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Why SHO in safflower in Australia?
• Oleic acid is also the starting substrate for other valuable industrial oils and waxes
• Sustainable/renewable bio-economy is a strategic goal for an Australian bio-economy
• Bio-based production of materials and chemicals
• Sustainable plastics from ‘fresh’ oils
• GM-proteins
• Antibodies
• Silk proteins
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High oleic safflower
• Low oleic safflower contains about 20% oleic acid
• High-oleic safflower lines have around 75% oleic acid
• Remainder is linoleic (18:2) and saturates (16:0)
• What genes are changed in safflower to create high oleic oil?
• What genes remain unchanged that could contribute to the remaining linoleic acid?
70 20 10 Low Oleic
15 75 High Oleic
Linoleic Oleic Saturates Linolenic
10
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Molecular basis of high oleic safflower
• Extensive EST library screening in seeds and vegetative tissue of both low oleic and high oleic genotypes
• Deep sequencing of RNA populations in low oleic and high oleic genotypes
• Biochemical characterisation of candidate genes
70 20 10 Low Oleic
15 75 High Oleic
Linoleic Oleic Saturates Linolenic
10
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Gene expression analysis in developing seed of safflower
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At least 11 FAD2 genes in safflower confirmed using southern blots
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Only 3 safflower FAD2 are 18:1 desaturases Others have divergent activities
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Only FAD2-1 expression different confirmed using qRT-PCR
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Molecular basis of high oleic safflower
• FAD2-1 genes were sequenced from low oleic safflower and three high oleic varieties (S-317, LeSaf-496 & CW99-OL).
5’ 3’
HIS-1 HIS-3 HIS-2 Low Oleic FAD2-1
HIS-1 HIS-2 High Oleic fad2-1
• All three high oleic varieties had the same mutation in fad2.1
• High oleic safflower first described by Horowitz and Winter (Nature, 1957)
380 aa
Frame shift mutation and dysfunctional 18:1 desaturase
Functional 18:1 desaturase
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Can super-high levels of oleic acid be produced by silencing CtFAD2-2? and CtFATB?
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• Large family of FAD2 in safflower • Some expression in seeds is detectable
• Some cross-silencing allowed for in this hairpin design
RNAi-based silencing FAD2-2 and FATB genes in safflower seed
AtOleosin FATB FAD2-2
FATB FAD2-2
hpRNAi-CtFAD2.2-CtFATB
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• Specialist technique developed for safflower genetic transformation • Belide et al 2011 (Plant Methods)
• GM shoots are rescued via micro-grafting
• 40 independent lines generated
• Data for elite lines presented here • Mature T3 seed
High oleic safflower transformed with seed-specific hpRNAi-CtFAD2.2-CtFATB
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Increasing 18:1 content from 78% to 94%
18:0
16:0
18:1
18:2
HO safflower
SHO safflower
Fatty acid - % of total
18:1 78
18:2 15
16:0 7
18:0 1
Fatty acid - % of total
18:1 94.5
18:2 2.5
16:0 2.0
18:0 1
HO safflower +hpRNAi
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High-oleic oils Where does SHO safflower oil rank?
94% SHO Safflower
Linoleic Oleic Saturates Linolenic
HO Soybean 77%
HO Canola 80%
82% HO Sunflower
• Highest oleic
• Lowest linoleic
• Lowest saturates
• Zero linolenic
75% HO Safflower
RA 80% HO Castor
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Molecular basis of high and super high oleic acid oils in safflower • Safflower has a large number of FAD2 genes
• Some are classic 18:1 desaturases
• FAD2-1 is mutated in high oleic safflower varieties
• Seed-specific silencing of CtFAD2.2 and CtFATB results in over 94% oleic acid in safflower
Super high oleic safflower trait developed
No developmental drawbacks yet seen in SHO lines…..
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Industrial products derived from SHO High value industrial traits
SHO 18:1
RA
DHSA
– C – C – C –
OH
–
– C – C –
C
hydroxylated
cyclic
WAX
ERUCIC
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Thank you
CSIRO Plant Industry
Craig Wood
t + 61 2 6246 5065 e [email protected]
ISPL2012 SEVILLE SPAIN
Qing Liu Shijiang Cao Xue-Rong Zhou Stuart Stephan Surinder Singh Allan Green