Download Increasing the vitamin E content in plants by overexpressing the γ

Increasing the vitamin E content
in plants by overexpressing the γtocopherol methyltransferase
gene
Craig Schenck
11/12/09
Vitamin E
• Collective term for α-,β-,γ-,δ-tocopherols
and four corresponding derivatives α-,β-,γ,δ-tocotrienols
• α-tocopherol is the most active form of
vitamin E and is considered the most
important for human health
• Tocopherols are synthesized exclusively in
photosynthetic organisms
Health Benefits for Humans
• Vitamin E is a strong antioxidant
• Vitamin E may also be implicated in the
prevention of heart disease, cancer, loss
of memory and eye disorders
• Deficiencies in vitamin E cause poor
transmission of nerve impulses, muscle
weakness and vision degradation
Sources of Vitamin E
Food
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Serving
Milligrams
% RDA
Egg, whole, fresh .................................... 1 large ........................................0.88 ......................................5.8
Almond oil ..........................................1 tablespoon ....................................5.3 ......................................35.3
Corn oil (Mazola) ................................1 tablespoon ..................................... 3 .......................................... 5
Cottonseed oil .....................................1 tablespoon ....................................4.8 ....................................... 32
Olive oil ..............................................1 tablespoon ....................................1.6 ......................................10.6
Peanut oil ............................................1 tablespoon ....................................1.6 ......................................10.6
Soybean oil ..........................................1 tablespoon ....................................1.5 ....................................... 10
Vegetable-oil spray .......................... 2.5 second spray ................................0.51 ......................................3.4
Wheat-germ oil ...................................1 tablespoon ...................................20.3 ....................................135.3
Apple with skin .....................................1 medium .....................................0.81 ......................................5.4
Spaghetti pasta, enriched ......................... 1 cup .........................................1.03 ......................................6.8
Almonds, dried ....................................... 1 ounce .......................................6.72 .....................................44.8
Hazelnuts, dried ..................................... 1 ounce ........................................6.7 ......................................44.6
Peanut butter (Skippy) ........................1 tablespoon ..................................... 3 .......................................... 5
Peanuts, dried ......................................... 1 ounce .......................................2.56 ...................................... 17
Margarine (Mazola) ............................1 tablespoon ..................................... 8 ........................................53.3
Mayonnaise (Hellmann’s) ...................1 tablespoon .................................... 11 .......................................73.3
Spinach, raw ............................................1/2 cup .......................................0.53 ......................................3.5
Sweet potato ..........................................1 medium .....................................5.93 .....................................39.5
Tomato, red, raw ....................................1 tomato ......................................0.42 ......................................2.8
Tocopherol biosynthesis
The tocopherol biosynthetic pathway and mutants in Arabidopsis thaliana and Synechocystis sp. PCC6803. Enzymes are indicated by black
boxes and mutations by red letters and lines. Bold arrows show the primary biosynthetic route in wild-type Arabidopsis leaves and Synechocystis.
vte1, vte2, vte3, vte4 and pds1 are mutants that have defects in tocopherol cyclase (TC), homogentisic acid (HGA) phytyltransferase (HPT),
2-methyl-6-phytyl-1,4-benzoquinol (MPBQ) methyltransferase (MT), g-tocopherol methyltransferase (g-TMT) and hydroxyphenylpyruvate (HPP)
dioxygenase (HPPD) in Arabidopsis, respectively. slr1737, slr1736, sll0418, slr0089 and slr0090 are the corresponding mutants in Synechocystis.
Null alleles of Arabidopsis pds1 and vte3 are lethal because of plastoquinone (PQ) deficiency [28,41], whereas the disruption of slr0090 or sll0418
has no impact on PQ and viability in Synechocystis [28,42]. DMPBQ, 2,3-dimethyl-6-phytyl-1,4-benzoquinol; PDP, phytyl-diphosphate; SDP,
solanesyl-diphosphate.
Soybean and perilla
Soybean
Perilla
Methodology
• Soybean transformation
– Particle bombardment via gene gun
– Shot gold/DNA coated microprojectiles into
embryogenic cultures
– Cell culture work, lasting approximately 7 months
Schematic representation of the binary vector constructed for soybean transformation. The T-DNA region of the
p1304PfTMT binary vector showing the assembly of γ -TMT expression cassette (Vicilin P:γ -TMT:OCS T), hygromycin
expression cassette (35S P:HYG:35Spoly A) and GFP–GUS reporter fusion gene expression cassette (35SP:GFP–
GUS:Nos poly A). γ -TMT: γ -tocopherol methyltransferase;OCS T: Agrobacterium tumefaciens octopine synthase poly A;
35SP: CaMV 35S promoter; 35S poly A: CaMV 35S poly A; HYG: hygromycin;GFP–GUS: green fluorescent protein – βglucronidase fusiongene; Nos poly A: Agrobacterium tumefaciens nopaline synthasepoly A
Methodology
• Perilla transformation
– Similar construct to soybean transformation
however Agrobaterium-mediated
transformation was used
pBKI
Figure 1. Schematic representation of T-DNA region of binary vectors pIG121Hm (top) and
pBK I (bottom). LB, left border; RB, right border; NOS PRO, nopaline synthase promoter;
NPT II, neomycin phosphotransferase II; NOS TER, nopaline synthase gene terminator;
35S PRO, cauliflower mosaic virus 35S promoter; GUS, b-glucuronidase gene; HPT,
hygromycin phosphotransferase; 35S poly A, 35S terminator; BAR, gene encoding
phosphinothricin acetyl transferase; Vic PRO, vicillin promoter; OCS, octopinesynthase
terminator.
Identifying transgenic lines and
gene expression of soybeans
Northern blot of RNA isolated from different tissues
of wild type and T134 plant
Southern blot probed with full length GUS gene
Transgenic perilla lines
(b) PCR amplification
of transformed γ-TMT gene. M, molecular size
marker P, plasmid (pBK I) as a positive control; Y,
Yeupsil as
a negative control; P1–P4, transgenic T0 plants
recovered from different transformation events; P1-1, a
T1 progeny of P1 that does not carry the transgene due
to segregation; P1-2 and P1-3, T1 progenies of P1
carrying the transgene.
Southern blot of T2 plants
Analyzing progeny and expression
pattern
- Northern blot analysis confirms
seed specific expression in both
T0 and T1 generation
- γ-TMT gene is heritable
Southern and northern blot analysis of T1 progeny probed with
full length γ-TMT gene
Expression pattern of γ-TMT
gene
-γ-TMT gene is expressed only in the
immature seed
-The gene is heritable through multiple
generations
Fig. 2 Northern blot analysis of transformed c-TMT gene
transcript. Upper panel: Northern blot. lane 1, Yeupsil leaf;
lane 2, T0 plant (P2 leaf); lane 3, Yeupsil (immature seed)
10 days after flowering; lane 4–7, Immature seeds of P1-2, P13, P2-1, and P2-2, respectively, 10 days after flowering. Lower
panel: ethidium-bromide gel staining of RNA samples as
loading control
Tocopherol content in seeds of
soybeans
Tocopherol content in seeds of
perilla
Conclusion
• Successfully transformed γ-TMT gene in both
cases
• The trait is heritable across multiple generations
• α-tocoperol content increased 10-20
• Manipulation of vitamin E synthesis did not
significantly impact total lipid contents and fatty
acid compositions of the seeds.
• Agrobaterium-mediated transformation more
efficient than particle bombardment?
References
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Tavva VK, Kim YH, Kagan IA, Dinkins RD, Kim KH, Collins GB (2007) Increased αtocopherol content in soybean seed overexpressing the Perilla frutescens γtocopherol methyltransferase gene. Plant Cell Rep 26:61–70.
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Lee BK, Kim SL, Kim KH, Yu SH, Lee SC, Zhang Z, Kim MS, Park HM, Lee JY (2008)
Seed specific expression of perilla γ-tocopherol methyltransferase gene increases αtocopherol content in transgenic perilla (Perilla frutescens). Plant Cell Tissue and
Organ Culture 92:47-54.
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Maeda H, DellaPenna D (2007) Tocopherol functions in photosynthetic organisms.
Current Opinion in Plant biology 10:260-265.
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Department of Human Nutrition, "Vitamin E." November
2004.http://ohioline.osu.edu/hyg-fact/5000/5554.html (accessed 11/8/09).
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Office of Dietary Supplements, "Vitamin E Fact Sheet." October 4,
2007.http://ods.od.nih.gov/factsheets/vitamine.asp#en4 (accessed 11/8/09).