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COCONUT (Cocos
nucifera L.)
1
Implementing and Funding
Agency: UPLB-PCARRD-
DOST
Source of Fund: 89-734-23
Total Budget: P1,536,274.96
Duration: Phase 2, 3 years
Objectives
General:
To clone and determine all possible
isoforms of all the major genes
involved in fatty acid metabolism
and to transform the coconut
thioesterase gene in corn
2
Objectives cont…
Specific:
Study 1: To clone isoforms of the acetyl
CoA carboxylase (ACCase),
phosphatidic acid phosphatase (PAP),
βketoacyl (ACP) synthase 3 (KAS 3),
and acyl-ACP thioesterase (TE), acyl
carrier protein (ACP), lysophosphatidic
and glycerol-3-phosphatase
dehydrogenase genes.
Objectives cont…
Study 2: To clone lysophosphatidic acid
acyltransferase (LPAAT) gene from
coconut
Study 3: To over express and transform
the coconut thioesterase gene in corn
Study 4: To perform a coconut promoter
analysis
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Coconut (Cocos nucifera
L.) is the major export crop
of the Philippines
Widely disbributed in
approximately 11.6 ha. in
86 countries
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PART I
CLONING OF ISOFORMS OF
ACCase, PAP, KAS 3, TE,
ACP, LPAAT GENES FOR
FATTY ACID METABOLISM
Coconut Oil
naturally saturated
vegetable oil
has food, industrial
and medical
purposes
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USES OF COCONUT OIL AND ITS DERIVATIVES
Edible Uses
Refining
Hydrogenation
..Frying Oil
..Cooking Oil
Shortening
..Margarine
Vanasoati Soap Stocks
..Filled Milk
Filled Cheese
..Ice Cream Coating
Meilorines Acid Oil
..Coffee Whitener
Whipped Dessert Topping
..Confectionery
Spray Oil (Cereals, Cookies, Crackes)
Salad Oil
Fatty Acid Soap
KAS III
4:0 - ACP
TE
TAG
C16-C18 - ACP
G-3-P
18:1 - C16-C18-CoA PA
ACP
Galactolipids PAP
Phospholipids
Plastid
Endoplasmic
Reticulum
(Ohlrogge & Jaworski, 1997) DAG
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METHODOLOGY
mRNA Isolation
4,5 and 6 month old
4 5 6
3’ RACE
(Rapid Amplification of
cDNA ends)
4 5 6
Primer Design
Primer Sequence Source Molecular %GC
Name Weight
TE-S NQHVNN Jones et 6415.8 42
al,1995
(AAYCARCAYGTNAAYAAY)
ACCase-S EVEVMKM Podkowinski et 6591.8 40
al,1996
(GARGTNGARGTNATGAAR)
LPAAT-S FPEGTRS Knutzon et al, 5501.3 61
1995
(TTYCCNGARGGNACNCGN)
ACP-S DTVEI Suh et al.,1999 5618.4 43.5
(GAYACNGTNGARATHGTN)
PAP-S DDYWH Marcel et 5537.1 41.7
al.,2000
(GAYGAYTAYTAGCAYCAY)
KAS3-S DITAA Tai & 4654.2 62.2
Jaworski,1993
(SWRCANGCNGCNGTD)
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ONTOGENETIC EXPRESSION & ISOFORM DISCOVERY
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Phosphatidic Acid Phosphatase (PAP)
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Lysophosphatidic Acid Acyl
Transferase (LPAAT)
4 5 6
Using the ACCase-S as
forward primer
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)
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NOTE!!!
The mRNA transcript or the gene of
interest are found at varying ages
Suggests ONTOGENETIC
EXPRESSION…
Ontogenetic Expression
Genes Age of Coconut Endosperm
4 5 6
ACCase
ACP
KAS 3
TE
LPAAT
PAP
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suggests enzymatic and gene
regulation occur early in the
endosperm
Isoform Discovery
Both PAP and KAS 3 genes were found in one
plant species, the coconut
PAP:castor beans, Phaseoulus vulgaris
and safflower seeds
KAS 3: E. coli and Spinach
The results obtained provides the first evidence
of the presence of the KAS 3 gene in a woody
monocot such as the coconut
The 8 isoforms of the ACCase gene found in
coconut is unique since only 7 known isoforms of
the ACCase gene has been published.
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GENE DISCOVERY
GAGGTTGAGGTAATGAAGATGCGCGCTCGCTAT
TTTCTTTTGCCAGTGGTTGGTCTGTGCTTTTTT
GTCTAAATAACATTGTACAAGATGGTTCAATATT
CGACAGCATGATTTGAACTTTTGAAAAAAAAAA
AAAAAAAA
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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
5 4
compare both
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Sequence Analysis
TE(2)-5 vs. TE-LIB TE(2)-6 vs. TE-LIB
Homology Homology
1.0 1.0
0 0
1 51 101 151 201 251 301 351 401 451 501 551 601 651 701 751 794 1 51 101 151 201 251 301 351 401 451 501 551 601 651 701 751 794
Homopolymer
T-chain
Poly-A tail
Representative sequence
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The three coconut PAP genes were cloned
GAC ATC ACT GCA GCG TGC AAG GAG ACC AAG GCC CGC TTC GAG
GAG CGC TTC AAG ACC GGC AAG AAC CGG TGG TTC TTC ACT AAG
CTC CGC TTC TAG ATA AAG GTA TAC AGT TCA TGA GAT TGA TGC CCG
CTG TCT AGG GTT TCT TTC TTA TAT TGT TAT GGA TCG CTT TGC TTA
GTC GTC GTC GTT GCT ATG GTT GGT GTC ATT TGG ATT TGT TAT TGG
ATC TAG TAG ACG TTT TAA TGT AGT TGG CAC TTA ACT GTT TTG ATG
GAG TTA CTT GGT TAT ATG TTT AGG ATG AATGGA TCC GGA TGA GTT
CAA TAA AAA AAA AAA AAA AAA AA
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Conserved region
D I T A A C S G F
Active-site cysteine
ACTIVE-SITE CYSTEINE
1. Suggested to be involved in substrate-binding,
2. Conformational changes during catalysis, and
3. Acid-base catalytic mechanism
---Qui et al., 1999
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Coconut ACCase gene
GAGGTTGAGGTAATGAAGATGCGCGCTCGCTATTT
TCTTTTGCCAGTGGTTGGTCTGTGCTTTTTTGTCTA
AATAACATTGTACAAGATGGTTCAATATTCGACAGC
ATGATTTGAACTTTTGAAAAAAAAAAAAAAAAAA
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MOLECULAR-BASED COCONUT (MC)
STANDARD
COCONUT TISSUE CULTURE
for successful coconut
tissue culture to occur,
genes that are responsible
for medium-chain fatty acid
should be present in the
starting material (Lopez-
Villalobos,2001)
using the MC
Standard,detection of the
genes involved in
medium-chain fatty acid
(C12:0 or lauric acid)
synthesis can be readily
made.
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VCO STANDARDIZATION
PART II
ONTOGENETIC PATTERN OF
DOUBLE-STRANDED FREE
NUCLEIC ACID (DS-FNA) FROM
THE WATER OF COCONUT
(COCOS NUCIFERA L.)
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Coconut Water (CW)
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Methodology
Extraction of DS-FNA at
Establishing presence of varying ages
DS-FNA in CW (LR 14-22)
(GFX™ Purification Kit, (DNA Binding Matrix,
Amersham) Eppendorf)
Crude Concentrated
Colorimetric Analysis
(DNA Dipstick, Invitrogen)
LR 15 (Pre-endosperm
development)
LR 20 (Late endosperm (+) (+)
development) A B
LR 20
DS-FNA is present!!!
(+) (-)
This is interesting!!!
A) Control (Purified CW
compliments of Guzman)
Does this mean that DS-FNA
B) Purified CW using GFX™
presence depends on its age? Purification Kit (Amersham)
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II. Extraction of DS-FNA in CW at varying ages
A. Crude CW LR 14,15,16
DS-FNA
0.0 ng/μL
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B. Concentrated CW LR 14,15,16
Results show…
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Estimated concentration
9
8
7
(ng/uL)
6
5
4
3
2
1
0
14 15 16 17 18 19 20 21 22
Leaf rank
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ACKNOWLEDGMENTS:
THANK YOU!!!
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