Download 1 Objectives

Coconut Biotechnology:
Gene Discovery of Fatty Acid/
Triglyceride Biosynthesis,
Cocosin Promoter and
Tissue Culture-Transformation
in Corn as a Model System
Rita P. Laude
 Coconut (Cocos nucifera L.)
is the major export crop of
the Philippines
 Widely distributed in
approximately 3.26 M has.
in 68 provinces with ~ 12 B
nuts
 Average annual contribution
is US$850M (>1% of total
(GDP)
 Of key importance is its
lauric oil content (48-57%)
which commands premium
price in world markets
(Banzon and Velasco, 1982)
USES OF COCONUT OIL AND ITS DERIVATIVES
Edible Uses
Refining
Hydrogenation
Frying Oil
...Cooking
Oil
Shortening
Vanasoati
...Margarine
Filled Milk
..Filled
Cheese
Ice Cream Coating
Meilorines
...Coffee
Whitener
Whipped Dessert Topping
..Spray
Oil (Cereals, Cookies, Crackes)
.Confectionery
Salad Oil
Soap Stocks
Acid Oil
Fatty Acid
Soap
Non Edible Uses
Saponification
Hydrolysis
Ethoxylation
Interesterification
Hydrogenation
Sulfation
Amidation
Reaction with alkalis, carbonates, amines, oxides
..Laundry
Soap14. Lubricant for Textile and Leather
Toilet Soap15. Viscosity Modifiers for Lube Oil
..Fuel
Extender16. Plasticizers for Plastic
Surfactants for Synthetic Detergent17. Synthetic Resins
..Shampoos/Bubble
Bath/ Shower Bath18. Paper Processing
Cosmetics/Toiletries19. Paints, Varnishes and Inks
Surfactants for Enhanced Oil Recovery20. Pharmaceuticals
...Foam
Boosters21. Anti-sucker Agent
Industrial Cleaning Applications22. Toothpaste
Emulsifier for Agricultural Chemical23. Explosives
...Emulsifier
in Oil Drilling24. Humectants
.Tin Plating25. Suppositories
Diesel Oil Substitute
 Coconut Oil
naturally saturated
vegetable oil
has food, industrial
and medical
applications
Coconut oil can also contribute
to energy demands of the country
aside from food, health and
industry needs.
Demand potential for
coconut will increase for
food & health applications,
industry and as a renewable
energy source
A highly productive coconut
with dramatic increase in oil
yield is needed;
Development of coconut
with high oil content can be
achieved through over
expression of genes for fatty
acid and triglyceride
biosynthesis
Objectives
General:
To develop a practical functional
gene analysis system for the future
coconut of the Philippines with
significant increase in oil content
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Specific Objectives
To isolate and characterize the major
genes in fatty acid and triglyceride
biosynthesis
For fatty acid biosynthesis: (4 genes)
acetyl CoA carboxylase (ACCase)
beta-ketoacyl (ACP) synthase 3 (KAS3)
acyl-ACP thioesterase (TE)
acyl carrier protein (ACP)
For triglyceride biosynthesis: (2 genes)
lysophosphatidic acid acyl transferase (LPAAT)
phosphatidic acid phosphatase (PAP)
Fatty Acid/Triglyceride Biosynthesis
Specific Objectives
To isolate and clone a strong
endosperm-specific promoter that can
overexpress genes in corn and in the
future, coconut
To develop a transformation system in
corn as “proof-of-concept” tool for the
isolated and characterized coconut
genes
Key enzymes in fatty acid biosynthesis
Cloning of Key Coconut Genes
in Fatty Acid and Triglyceride
Biosynthesis:
ACCase, ACP, KAS 3, TE, PAP
and LPAAT
METHODOLOGY
mRNA Isolation
4,5 and 6 month old
C6H12O6
Oil bodies in coconut
endosperm
ACCase
ACP
malonylacetylmalonyl
ACP
CoA
-CoA
ACCase
4
TAG (oil)
KAS III
4:0 - ACP
TE
18:1 ACP
Plastid
TE
KAS3
G-3-P
C16-C18-CoA
LPAAT
and
PAP
PA
Phospholipids
Endoplasmic
Reticulum
5
6
3’ RACE
(Rapid Amplification of
cDNA ends)
ACP
C12
C16-C18 - ACP
Cytoplasm
TE
Fatty acid synthase
complex
To increase fatty acid biosynthesis, there is a need
to overexpress these 4 key enzymes in the
fatty acid synthase complex.
4
Agarose Gel Electrophoresis
5
6
TOPO TA Cloning
(Ohlrogge & Jaworski, 1997)
2
RESEARCH HIGHLIGHTS
Ontogenetic and Isoform Expression
Genes
Age of Coconut Endosperm
4-mo
FA
TAG
ACCase
2
ACP
1
5-mo
6-mo
8
KAS 3
2
2
TE
2
1
LPAAT
2
PAP
3
-ketoacyl (ACP) synthase 3 (KAS 3)
 Using the KAS3-S
primer as forward
primer
 Two isoforms (725 bp
and 425 bp sizes)
were found from 5and 6- mo. old
coconut endosperm
 Similar with the works
of Jaworski et al,1989
& Dehesh et al.,2001
ONTOGENETIC EXPRESSION & ISOFORM DISCOVERY
Acyl Carrier Protein
(ACP)
 Done using COLD-START PCR
 Using the ACP-S primer as
forward primer, a 300 bp PCR
band was generated in the 4-mo.
old coconut endosperm
 No band was generated from the
5- and 6- mo. old tissues
Isoform 1 (725 bp)
Isoform 2 (425 bp)
4
ACP
5 6
TOUCHDOWN PCR
 No PCR bands from
total RNA of 4-mo. old
coconut endosperm
 Two PCR bands from
5-mo. old coconut
endosperm
 Only isoform 2 (460
bp) was observed
from 6-mo. old
coconut endosperm
5
4
forward primer
5
6
 Eight isoforms were
found at the 6 mo. old
cDNA
 Two isoforms were
found at the 4 mo. old
cDNA
 No bands were present
in the 5 mo. old coconut
endosperm
 Similar with the works of
Podkowinski et al.
(1996)
Acyl-ACP thioesterase (TE)
 HOTSTART +
4 5 6
Acetyl CoA carboxylase (ACCase)
 Using the ACCase-S as
Phosphatidic Acid Phosphatase (PAP)
 Using the PAP-S primer
4
as forward primer
4 5 6
 Three isoforms (700bp,
Isoform 1 (600bp)
Isoform 2 (460bp)
6
500bp and 400bp sizes)
were found from 6-mo.
old coconut endosperm
 Similar with the works of
Moore et al.(1973),
Paliyath & Thompson
(1987) and Styme et al.
(1993).
Isoform 1 (700 bp)
Isoform 2 (500 bp)
Isoform 3 (400 bp)
Isoform 2 (460bp)
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Plastid
Lysophosphatidic Acid Acyl
Transferase (LPAAT)
Cytoplasm
C2
ACCase
C4
 Using the LPAAT-S
primer as forward
primer
 Two isoforms (700bp
and 400bp) detected
from 6-mo. old
coconut endosperm
 Similar with the works
of Sun et al (1988) and
Davies et al.(1995)
4
5
Ontogenetics and Isoform Discovery
in the Developing Coconut Endosperm
2001000
FFA1
6
300
KAS3
ACP
725
425
FFA2
glycerol
TAGs/oil
FFA3
Isoform 1 (700bp)
Isoform 2 (400bp)
C8
C10
C12
C14
C16
C18
TE
600
460
Free fatty
acids
FFA LPAAT
PAP
700
500
400
Triglycerides
700
400
Storage into
Lipid Bodies
The presence/absence of mRNAs and
multiple forms of the same mRNAs of
the 6 major genes in fatty acid and
triglyceride biosynthesis reflect fine
and coordinated gene regulation
between fatty acid biosynthesis in the
plastids and triglyceride biosynthesis
in the cytoplasm.
Cloning the coconut ACCase gene
GENE DISCOVERY: DNA Sequencing
Cloning made use of
the Topo TA Cloning
kit (Invitrogen)
Selected genes
were cloned,
namely: ACCase
(from 4 mo.); TE
(from both 5 and 6
mo.) and PAP (from
6 mo.)
GAGGTTGAGGTAATGAAGATGCGCGCTCGCTAT
TTTCTTTTGCCAGTGGTTGGTCTGTGCTTTTTT
GTCTAAATAACATTGTACAAGATGGTTCAATATT
CGACAGCATGATTTGAACTTTTGAAAAAAAAAA
AAAAAAAA
Features of Raw sequence data:
 contains 140 nucleotides
 primer location:1-21(red)
 poly A tail: 124-140
termination signal (green)
possible adenylation signal (underlined)
Features of all ACCase Genes
 EVMKM conserved region ( 7-21)
 MKM motif -biotin binding site (13-21)
 Proline (CCA) residue -forms a hinge region for carboxybiotin
movement(43-45)
 Lysine (GAA) residue- highly conserved region (17-19)
Conclusion: NOVEL coconut ACCase gene was cloned from the 4 mo.
old coconut endosperm
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Coconut Promoter
The promoter of the cocosin gene is strong
due to high levels of the protein and
expression is specific only in the endosperm.
Why do we need a promoter?
Methodology
PCR-based Cloning of Cocosin Promoter
gDNA coconut
1. A strong promoter duplicated in tandem can
overexpress any gene
2. The 35S CaMV promoter is a strong promoter
but patented by Monsanto
3. The 35S CaMV promoter is constitutive and
will overexpress any gene in all tissue parts
4. The level of expression is generally higher
in dicots compared to monocots
PCR with designed primers
based on cocosin gene
200 bp PCR product
DNA sequence analysis
vector insertion
pCAMBIA expression
with gus gene
Transient Expression
Gene Construct
PCR-Based Cloning of the Cocosin Promoter
1. Maxi-prep of gene construct
2. Preparation of tissue culture materials
Papaya
zygotic
embryos
Negative control:
Tungsten only
Negative control:
Promoterless pBI
Positive control:
35S CaMVp pBI121
Cocosin promoter
3. Main transformation protocol
300 bp cocosin promoter
cocosinP
gus
nosT
1.3 kb fragment
Gus = beta-glucuronidase (reporter gene)
coconut endosperm
4. Reporter gene expression
5. Photodocumentation
Gene gun
using
tungsten
bullets
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Coconut Promoter Analysis
Stable transformation of coconut
thioesterase in corn
Research Highlights
I. Optimization of tissue culture conditions
II. Optimization of selection procedures
for transformation
III. Optimization of transient expression of
a reporter gene
Cloning and characterization of the
cocosin promoter
Preparation of gene construct for
functional analysis
RESEARCH HIGHLIGHTS
1.
Developed and optimized tissue
culture system for callus initiation,
somatic embryogenesis and plantlet
regeneration
First coconut promoter to be isolated,
characterized and proven functional
Developed and optimized tissue culture conditions in corn.
Optimization of somatic embryogenesis …
Highlights of Accomplishments…
2.
10-11 day-old cob
Differentiation of embryogenic
calli
a
Optimized protocol for successful ex
vitro establishment of regenerated plants
b
Plantlet regeneration
Root emergence, callus
initiation
Germination of somatic
embryos
Ex vitro plant
establishment
Detailing (removal of
emerged roots)
Formation & proliferation
of friable calli
Acclimatization of corn plants.
c
Shoot proliferation
Fertile corn plant derived from
tissue cultured maize
Somatic embryogenesis and plantlet regeneration in white corn inbreds.
d
Callus induction with N6 basal medium supplemented
with 2 mg/L Dicamba (a); and subsequent plantlet
regeneration of P53 inbred (c-d).
Regenerated plants establishment in the soil.
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Highlights of Accomplishments…
Highlights of Accomplishments…
3.
The survival of regenerated plants ex vitro is
affected by the quality of the plants produced
in vitro.
Plantlets with healthy, well-developed shoots
and roots showed higher survival during
transfer to soil.
Tissue culture protocol developed
produced normal and fertile tissue
cultured corn plants
Tassel
Ear
Highlights of Accomplishments…
4.
Optimized selection procedures and
identified good selective agents for
transformed cells
 The antibiotic kanamycin and herbicide PPT as
selective agents were not appropriate for corn
transformation.
 Untransformed corn tissue cultures at all stages of
selection (callus stage, 2- regeneration stage, rooting
stage) were more sensitive to geneticin than
kanamycin.
Survival of the transplanted corn
plants to
soil is greatly affected by environmental
conditions.
Normal and fertile corn plant regenerated from in vitro culture.
 Geneticin is a better selective agent for selection
of transformed cells (No false positives)
For example…
Highlights of Accomplishments…
5. Optimized transient transformation
A
Transient expression of the
reporter GUS gene detected in
both particle inflow gun (biolistic
method) and Agrobacterium
mediated transformed tissues of
corn
C
To summarize the 4 studies…
B
D
Gus expression in 5-day old
pre-cultured callus (A,C)/
embryo (B, D)
Gene discovery
endosperm-specific
for oil biosynthesis promoter to regulate
(6 genes)
gene expression
Transformation
System in Corn
•
To increase oil content, need to increase
ACCase activity
•
To increase lauric acid content, need to
increase thioesterase activity
•
To increase free fatty acid incorporation
to glycerol, need to increase LPAAT
or PAP activity
Strategy is to prepare a gene construct
that contains all 3 genes, transfer into
corn and see what happens . Proof of
concept in 1.5-2.0 years
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Structural genes in coconut
(Dr. Laude’s studies)
Cocosin promoter
Dr. Laurena’s study
Tissue culture/transformation
in corn
(Dr. Olivia Damasco’s study)
To test efficacy of gene expression in corn
To observe phenotype in corn by overexpression
of coconut genes in oil biosynthesis
We believe that
“innovation and creativity”
generated by our research and
the emergence of new technologies
will initiate the next quantum leap
in coconut research.
Research
Team
Rita P. Laude, Ph. D.
Ma. Genaleen Q. Diaz, Ph. D.
Merlyn S. Mendioro, Ph. D.
Marni E. Cueno
Olivia P. Damasco, PhD.
Antonio C. Laurena, PhD.
Jorge Gil C. Angeles
Lucilla R. Laureles
Cleotilde A. Caldo
Institute of Biological Sciences
Institute of Plant Breeding (IPB)
University of the Philippines Los Baños
Philippine Council for Agriculture, Forestry and Natural
Resources Research and Development PCARRD Department of Science and Technology (DOST)
THANK YOU!!!
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