Advance Genetics

Genomic DNA Sequence Characterization of Pyruvate Decarboxylase in

Normal and Mutant "Makapuno" Coconut (Cocos Nucifera) Endosperm

Chale Noven Garcia Maravillas

Department of Biology, Central Mindanao University

chalenoven123@gmail.com

Abstract
This study aims to barcode the DNA of Pyruvate decarboxylase of normal and mutant coconut.
Specifically it aims to: (1)isolate the DNA of Pyruvate decarboxylase; (2) determine the DNA sequence
of Pyruvate decarboxylase; and (3)Compare the DNA sequence of normal and mutant coconut Pyruvate
decarboxylase. Makapuno and normal coconut endosperms were used as sample. CTAB-PVPP method
was used to extaract the DNA. Amplification of DNA sequence was done through polymerase chain
reaction. PCR products was visualized through gel electrophoresis. Two replicates from normal coconut
had PCR product. No PCR product was obtain in makapuno, so comparison between the DNA sequence
of normal coconut and makupuno cannot be determined. No bands on the DNA of makapuno. So further
PCR troubleshooting must be done to successfully amplify the DNA. Moreover, the DNA for pyruvate
decarboxylase must come from a fresh sample to ensure a higher concentration of DNA.

Keywords: genomic sequence, cocos nucifera, normal, mutant, makapuno

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Introduction

Matured coconut endosperm forms a hard kernel 0.5 to 1.5 cm thick ant it has cavity that

contains the residual nut water. There is a rare coconut endosperm mutation characterized by a

soft, extra-thick kernel, partially or completely filling the nut water cavity. This unusual mutation

affects only some of the fruit on the palm and such fruit will not normally germinate (Deva

Kumar, 2015). This mutated coconut was called Makapuno in the Philippines. In technical terms,

the macapuno is described as a ‘white viscous, translucent jelly’ (Ramirez & Mendoza 1998).

Makapuno was used in ice cream and macapuno meat is commonly processed into bottled

sweetened macapuno shreds (Lauzon, 2005).

Eight genes, namely two putative isoforms of α-galactosidase gene, cytosolic

glyceraldehyde-3-phosphate dehydrogenase, cytosolic pyruvate kinase, enolase), pyruvate

decarboxylase (Cnpdc) and two isoforms of ketoacyl-ACP synthase 1 (Cnkas 1-1 and Cnkas 1-2)

are involved in carbon metabolism in Makapuno. Gene from pyruvate decarboxylase was one of

the genes that uprgulated in Makapuno (Deva Kumar, 2015).

This study will isolate the genomic sequence of pyruvate decarboxylase. It is

hypothesized that the gDNA between the normal and mutant coconut will show a significant

difference.
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Objectives of the Study

This study aims to barcode the DNA of Pyruvate decarboxylase of normal and mutant

coconut.

Specifically it aims to:

1. isolate the DNA of Pyruvate decarboxylase;

2. determine the DNA sequence of Pyruvate decarboxylase; and

3. Compare the DNA sequence of normal and mutant coconut Pyruvate

decarboxylase.

Significance of the Study

Determining the gene responsible for the mutation in Makapuno has been going on for

decades. This study might give an essential clues that might explain the mutation of the

Makapuno

Scope and Limitations of the Study

The study was limited on the DNA sequence of pyruvate decarboxylase in the endosperm

of normal and mutant (makapuno) coconut.

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Genes, which were upregulated in the

Makapuno,
include Cnpdc, Cnpab, Cnmyb, Cnagal
1, Cnckb and Cnkas 1
Genes, which were upregulated in the
Makapuno,
include Cnpdc, Cnpab, Cnmyb, Cnagal
1, Cnckb and Cnkas 1

MATERIALS AND METHOD

The DNA extraction protocol

Endosperm from normal coconut and makapuno endosperm was used as biological

material for DNA extraction. Appropriate amount of CTAB isolation buffer was preheated in

65̊C water bath. 0.1 g of fresh samples was weighed and grinded. The samples were transferred

in a 2ml microfuge tube. 800 ul of CTAB isolation buffer was added to the samples and

incubated in 65̊C water bath for an hour.The tubes were then incubated at 65°C for 30 minutes

and inverted each 10 minutes for good solution of the crushed tissues with the buffer. The tubes
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will be cooled at room temperature and 800 uL of chloroform: isoamyl alcohol mixture (24:1)

was added. The tubes will be then mixed gently by inversion to form an emulsion during 10

minutes. After that the tubes will be centrifuged 6000 xg for 10 minutes. The aqueous phase

present in each tube (~500 uL) will be transferred to a new 2ml microcentrifuge tube gently, to

avoid DNA shearing. Tube will be added 600 uL of cold isopropanol (Stored previously in 65̊C).

the samples was mixed gently by inverting the tubes for several times. The solution will kept in

romm temperature overnight. Sample will be then centrifuged at 12,000 rpm for 1-2 minutes and

the supernatant was poured off. 800ul of ethanol was added to the DNA pellets and gently

swirled for 20 minutes. It was centrifuged for 3 minutes in 1200 rpm and the supernatant was

poured off. the pellet was air dried through inverting the tube to a paper towel. 100ul of High

Salt TE and 1ul of RNAse was added and was incubated for 30 minutes in 37̊C. The sample was

diluted with 200ul 1X TE10ul 10M sodium acetat (pH 5.2) and 250ul of 70% ETOH and the

DNA precipitate was mixed gently. The solution was centrifuged in 10,000xg for 10 minutes and

the supernatant was discarded. The sample was air dried until no traces of alcohol can be found.

Thhe sample was resuspend with 20ul TE buffer (10mM Tris-HCl, 1 mM EDTA (pH8)) to

dissolve the precipitate.

Polymerase chain reaction (PCR)

Polymerase chain reaction (PCR) will be carried out using the universal barcoding

primers PDC1 (GAT CAC TGG GAT TTG GTG) and reverse primer PDC2 (TCC TCA TAA

ACA ATG GGG G). PCR will be performed in a final volume of 15 uL. Each reaction

contained:

Table 1. First PCR Cocktail

Volume (uL)
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Ultra Pure Water 10.2

10X Standard Taq 10X 1X 1.5
Buffer
dNTPs 10uM 0.4uM 0.6
PDC1 (100 uM used) 10uM 0.2uM 0.3
PDC2 (100 uM used) 10uM 0.2uM 0.3
Taq Polymerase 5uM/uL 0.2uM 0.1
DNA (no dilution) 2.0
Total 15

Table 2.Second PCR Cocktail

Volume (uL)
Ultra Pure Water 10.1
10X Standard Taq 10X 1X 1.5
Buffer
dNTPs 10uM 0.4uM 0.6
PDC1 (100 uM used) 10uM 0.2uM 0.3
PDC2 (100 uM used) 10uM 0.2uM 0.3
Taq Polymerase 5uM/uL 0.2uM 0.2
DNA (2:50 dilution) 2.0
Total 15

Cycling conditions was: Pre-denaturation of 95̊C for 5 minutes and 30 cycles of

denaturation in 95̊C for 1 minutes. Annealing was in 55̊C for 30 seconds and elongation period

of 72̊C for 1 minute and 30 seconds. Then, the final extension was 72̊C for 5 minutes. PCR

blank controls was incorporated. PCR products were visualized on 1.5% agarose gels stained

with Gel Red (Biotium).

Genes, which were upregulated in the

Makapuno,
include Cnpdc, Cnpab, Cnmyb, Cnagal
1, Cnckb and Cnkas 1.
Advance Genetics

Genes, which were upregulated in the

Makapuno,
include Cnpdc, Cnpab, Cnmyb, Cnagal
1, Cnckb and Cnkas 1.
Genes, which were upregulated in the
Makapuno,
include Cnpdc, Cnpab, Cnmyb, Cnagal
1, Cnckb and Cnkas 1.
Genes, which were upregulated in the
Makapuno,
include Cnpdc, Cnpab, Cnmyb, Cnagal
1, Cnckb and Cnkas 1.
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RESULTS AND DISCUSSION

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Figure 2. First PCR Product of Normal Coconut and Makapuno Endosperm.

N1 N2 ML

Figure 2. Second PCR Product of Normal Coconut Endosperm and Makapuno Leaf. N1 (Normal
coconut replicate 1); N2 (Normal coconut replicate 2; ML (Makapuno Leaf).
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As shown in Figure 2 there were bands from the two replicates of normal coconut

endosperm but there were no bands for Makapuno endosperm. One reason might be the proper

proportion for diluting the DNA sample. It also had primer dimers because the concentration of

the primer was 100uM instead instead of using 10uM. If the primer concentration is in extreme

excess over the template concentration, then the primers will be more likely to anneal to

themselves or each other over the DNA template (Lorenz, 2012). According to Poritz and Ririe

(2014) high concentration of primers results to the formation of primer dimer

In Figure 1 showed no bands in the DNA. This might be because the DNA samples were

not diluted. Because after the samples were diluted in (2:50) ratio between DNA sample and

ultrapure water it showed bands in normal coconut indicating having a PCR product. Aside from

diluting the DNA, increasing the volume of Taq polymerase might have an effect on the the PCR

products in Figure 2. It may be necessary to increase the amount of Taq DNA Polymerase to 2-3

uL, if the PCR mixture contains inhibitors, for instance, due to contamination of the template

DNA (Thermo Scientific, 2012). This might also mean that the DNA samples do have

contamination. This contamination might be due to polysaccharides. Polysaccharides make the

DNA viscous, glue like and non-amplifiable during amplification in the PCR reaction. It inhibits

the activity of Taq polymerase and interferes with the restriction enzyme (Porebski et al., 1997).

There were no bands for makapuno endosperm it might be because the DNA was not

extracted or the DNA was low in concentration. According Lorenz (2012) the quality and

quantity of DNA may greatly benefit the outcome of a PCR experiment.

CONCLUSION
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Therefore, significant difference between the DNA sequence of Pyruvate decarboxylase

in normal and mutant coconut endosperm can’t be determined. Because the DNA of makapuno is

not yet sequenced and so comparison between their genome can’t be carried out. Further PCR

troubleshooting must be done to successfully amplify the DNA. Moreover, the DNA for

pyruvate decarboxylase must come from a fresh sample to ensure a higher concentration of

DNA.

REFERENCE

DEVA KUMAR, K., R. K. GAUTAM , I. AHMAD, S. DAM ROY and A.SHARMA.

2015.Biochemical, genetic and molecular basis of the novel and commercially
important soft endosperm Makapuno coconut - A review. Journal of Food, Agriculture
& Environment Vol.13 (1): 61-65. 2015
LAUZON R. D.2005. Physico-chemical properties and processing possibilities of macapuno
cultivars developed at leyte state university. Philippine Journal of Crop Science (PJCS)
August 2005, 30(2): 55-60 Copyright 2005, Crop Science Society of the Philippines
Released 19 June 2005
LORENZ, T.C. Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and
Optimization Strategies. Journal of Visualized Experiments : JoVE. 2012;(63):3998.
doi:10.3791/3998.

POREBSKI, S., L.G. BAILEY, B.R. BAUM. 1997.Modification of a CTAB DNA extraction
protocol for plants containing high polysaccharide and polyphenol components. Plant
Mol Biol Rep. 1997;15:8–15.

RAMIREZ, D.A. and E.M.T. MENDOZA. 1998. The Macapuno Mutant Coconut. Bicutan,
Taguig, Rizal: National Academy of Science & Technology
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THERMO SCIENTIFIC.2012. Components of the Reaction Mixture. Thermo Fisher Scientific

Inc. North America