Molecular Cloning and Characterization of Partial cDNA Encoding for Flavanone

3-hydroxylase from Kelampayan (Neolamarckia Cadamba)

LOW HUI CHIA

Bachelor of Science with Honours

(Biotechnology Resource)

2013

Faculty of Resource Science and Technology

Molecular Cloning and Characterization of Partial cDNA Encoding for Flavanone

3-hydroxylase from Kelampayan (Neolamarckia Cadamba)

Low Hui Chia

(26777)

This project is submitted in partial fulfillment of the requirements for the degree of

Bachelor of Science with Honours

Resource Biotechnology Programme

Department Of Molecular Biology

Faculty of Resource Science and Technology

University Malaysia Sarawak

2013

I

ACKNOWLEDGEMENTS

I would like to express my utmost gratitude to Dr. Ho Wei Seng for offering me the

opportunity to conduct this research project under his supervision. I learnt not only

laboratory skills and knowledge from him, but also the spirit of commitment and

perseverance towards scientific research. Besides, I want to thank my co-supervisor, Dr.

Pang Shek Ling for her patient guidance and professional advices along the journey of

this research. Also, thanks to Ms. Grace Ting (Msc. student) and Ms. Natalie Gali (Msc.

student) who have provided me aid and guidance throughout the project. I am also

indebted to all the labmates in Forest Genomics and Informatics Lab (fGil) and other

friends who have lent me help hands and being supportive all the way. Finally, special

thanks to my family who are always there for me through the ups-and-downs.

II

DECLARATION

I hereby declare that this thesis is of my original work except for quotations and citations,

all of which have been duly acknowledged. I also declare that it has not been previously

or concurrently submitted for any other degree at UNIMAS or any other institutions.

Low Hui Chia

Resource Biotechnology Programme

Department of Molecular Biology

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

III

TABLE OF CONTENTS

Acknowledgements I

Declaration II

Table of Contents III

List of Abbreviations V

List of Tables VI

List of Figures VI

Abstract/ Abstrak VIII

Introduction 1

Literature Review 3

2.1 Neolamarckia cadamba 3

2.2 Flavonoids in plants 5

2.3 Anthocyanin 6

2.4 Flavanone 3-hydroxylase 9

2.5 Flavanone 3-hydroxylase gene 9

Materials and Methods 11

3.1 Plant Materials Sampling 11

3.2 Total RNA Isolation 11

IV

3.3 Agarose Gel Electrophoresis 12

3.4 RNA Purity Determination and Quantification 13

3.5 Primer Designing 13

3.6 RT-PCR and Optimization of Polymerase

Chain Reaction (PCR) 14

3.7 Purification of PCR Product 16

3.8 DNA Sequencing and Data Analysis 16

Results and Discussion 17

4.1 Sample Collection and Total RNA Isolation 17

4.2 RNA Purity Determination and Quantification 19

4.3 RT-PCR and Optimization of Polymerase

Chain Reaction (PCR) 21

4.4 Gel Extraction 25

4.5 PCR Product Sequencing and Data Analysis 26

Conclusions and Recommendations 30

References 32

V

LIST OF ABBREVIATIONS

A Ampere

cDNA Complementary DNA

DNA Deoxyribonucleic acid

ddH2O Double-deionized distilled water

dNTP Deoxyribonucleotide triphosphate

LB Luria Bertani/Broth

MgCl2 Magnesium chloride

ml millimeter

µl micrometer

PCR Polymerase chain reaction

RNA Ribonucleic acid

rpm Revolution per minute

RT-PCR Reverse transcriptase - polymerase chain reaction

Ta Annealing temperature

Tm Melting temperature

X-Gal Bromo-chloro-indoyl-galactopyranoside

VI

LIST OF TABLES AND FIGURES

Figures Page

2.1 N. cadamba. (a) The tree of N. cadamba, (b) the leaves of N.

cadamba, and (c) the flower of N. cadamba.

4

2.2 The biosynthesis pathway of flavonoids. 6

2.3 The general structure of anthocyanin. 8

4.1 The agarose gel electrophoresis result of the total RNA

isolation from young leaves of N. cadamba using RLT buffer

(lane 2 and lane 4) and Plant RNA Isolation Reagent (lane 3

and lane 5). Lane 1: lambda hind III ladder marker, lane 2:

NMA, lane 3: NMB, lane 4: NCA, lane 5: NCB.

18

Figure 4.3 (a) The electrophoresis results showing the PCR product of

gradient PCR using cDNA as template at various annealing

temperatures. Lane 1: 100bp DNA marker ,lane 2: 34.9 °C,

lane 3: 35.2 °C, lane 4: 36.3 °C, lane 5: 38.0 °C, lane 6: 40.3

°C, lane 7: 42.9 °C, lane 8: 45.7 °C, lane 9: 48.4 °C, lane 10:

51.0 °C, lane 11: 53.2 °C.

24

Figure 4.3 (b) The electrophoresis results showing the re-amplified PCR

product of gradient PCR using initial PCR products as

template at various annealing temperatures. Lane 1: 100bp

DNA marker ,lane 2: 34.9 °C, lane 3: 35.2 °C, lane 4: 36.3 °C,

lane 5: 38.0 °C, lane 6: 40.3 °C, lane 7: 42.9 °C, lane 8: 45.7

°C, lane 9: 48.4 °C, lane 10: 51.0 °C, lane 11: 53.2 °C.

24

VII

Figure 4.4(a) The electrophoresis results of the PCR product prepared for gel

excision. 100µl of PCR product was loaded into the well of

lane 1, resulting in long double bands, while lane 2 shows the

100 bp ladder marker.

25

Figure 4.4(b) The electrophoresis results of the purified PCR product. Lane

1: 100 bp ladder marker, lane 2: lambda hind III, lane 3-5:

F3HS1 (~150 bp), lane 6 and 7: F3HS2 (~350 bp).

26

Figure 4.5 A middle section of the chromatogram of F3HS1. 27

Tables

Table 4.2 The RNA concentration and the readings of A260, A280,

A260/A280, A260/A230 from the isolated total RNA by using

nanodrop spectrophotometer.

20

Table 4.3 (a) The components and composition of the PCR reagents used in

the amplification of cDNA.

22

Table 4.3 (b) The PCR profile used in gradient PCR.

23

Table 4.5 Blastn results for the nucleotide sequence of F3HS1 28

VIII

MOLECULAR CLONING AND CHARACTERIZATION OF PARTIAL cDNA

ENCODING FOR FLAVANONE 3-HYDROXYLASE FROM KELAMPAYAN

(Neolamarckia Cadamba)

LOW HUI CHIA

Resource Biotechnology

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

ABSTRACT

Neolamarckia cadamba or Kelampayan is a type of deciduous tree species classified

under the Rubiaceae family. The fast fast-growing N. cadamba emerges as a new

alternative to meet high global demand of timber wood. Flavanone 3-hydroxylase is one

of the key enzymes involved in the flavonoid synthesis pathway. Thus, this study aims to

amplify the flavanone 3-hydroxylase partial cDNA in order characterize it through

sequence analysis. High quality total RNA was extracted from the young leaves of N.

cadamba and converted into cDNA. Amplification of cDNA encoding flavanone 3-

hydroxylase was performed through PCR. The BLASTn result of purified PCR product

revealed high similarity with the flavanone 3-hydroxylase (F3H) mRNA of other plant

species, comfirming the identity of the nucleotide sequence as the partial cDNA of

flavanone 3-hydroxylase.

Keywords: Neolamarckia cadamba, Flavanone 3-hydroxylase, RNA extraction, PCR,

sequence analysis

ABSTRAK

Neolamarckia cadamba atau Kelampayan ialah sejenis spesis pokok yang diklasifikasi di bawah

keluarga Rubiaceae. Tumbesaran N. cadamba yang cepat telah menjadikannya satu alternatif

baru untuk menampung permintaan kayu balak di dunia ini. Flavanone 3-hydroxylase merupakan

salah satu enzim yang amat penting dalam proses sintesis flavonoid. Oleh itu, objektif kajian ini

adalah untuk mengamplifikasi cDNA separa flavanone 3-hydroxylase untuk tujuan megenalpasti

jujukannya melalui analisa. Dalam kajian ini, RNA keseluruhan yang berkualiti tinggi telah

diekstrak daripada daun muda N. cadamba dan ditukarkan menjadi cDNA. Amplifikasi cDNA

flavanone 3-hydroxylase telah dilakukan dengan meggunakan PCR. Keputusan analisis BLASTn

daripada produk PCR menunjukkan persamaan yang tinggi dengan jujukan mRNA flavanone 3-

hydroxylase daripada spesis lain. Ini memastikan identiti jujukan tersebut sebagai cDNA separa

flavanone 3-hydroxylase.

Kata kunci: Neolamarckia cadamba, Flavanone 3-hydroxylase, Pengekstrakan RNA, PCR,

analisa jujukan

1

CHAPTER 1

INTRODUCTION

With rapid development and urbanization, forest trees rises in it economical importance

and also ecological values. Vast quantity of forest trees are logged for various uses in the

growth and development process. However, the timber supply can hardly cater the huge

demand over timber because of the slow growth rate of many wood species. This results

in the shortage of commercial timber supply and continual soaring of timber price.

Foreseeing the scarcity of wood supply in future, forest plantation of fast-growing wood

species would be the solution.

Neolamarckia cadamba, or locally known as Kelampayan, is a type of rapid-

growing deciduous tree classified under the Rubiaceae family. It has emerges as the new

alternative for the forest plantation to meet the high global demand of timber wood. N.

cadamba is widely known for its rapid growth rate and high quality wood properties,

which make it a good candidate as timber wood. N. cadamba is used for forest plantation

as the substitution of other timber species with slow growth rate. It is also a multipurpose

tree, which are widely applied as the material for plywood manufacturing, pulping,

flooring, furniture and crates production (Krisnawati, 2011). However, there are some

limitations of N. cadamba such as low adaptability, site-selective, and drought-intolerant,

which in turn affect the growth and development of N. cadamba. Kelampayan is the

indigenous species in South Asia and Southeast Asia countries such as like Malaysia,

Indonesia, Cambodia and India. Besides, it also prospers in Australia, Papua New Guinea

2

and China (Krisnawati et al., 2011). Apart from its commercial value, N. cadamba is also

reported to possess medicinal value such as anti-inflammatory and antidiabetic action

(Dubey et al., 2011).

The secondary metabolism is crucial for the survival and environmental fitness of

plants. One of the most abundant secondary metabolites in higher plants is the flavonoids.

Flavonoids have a variety of functions such as plant defense, antioxidant, disease

resistance, stress response and color determinant (Guo et al., 2008; Winkel-Shirley,

2001). Flavanone 3-hydroxylase is one of the key enzymes involved in the flavonoid

synthesis pathway. It catalyses the hydroxylation of flavanone into dihydroflavonal.

Flavanone 3-hydroxylase activity leads to the production of anthocyanin and flavonols.

Flavanone 3-hydroxylase is found to be strongly expressed during the secondary growth

of the tree. Thus, further research on the genes that manipulate the flavonoids metabolic

pathway will enhance adaptability of the tree. Therefore, the main aim of this study is to

gain a better understanding towards the genetic basis in N. cadamba to develop better

clones of Kelampayan in future.

The objectives of the project we\re to isolate high quality total RNA from young

leaves and developing xylem of Kelampayan tree, to amplify cDNA encoding for

flavanone 3-hydroxylase through RT-PCR with designed primers, and to characterize the

partial cDNA of flavanone 3-hydroxylase from Kelampayan through sequence analysis.

3

CHAPTER 2

LITERATURE REVIEW

2.1 Neolamarckia cadamba

Neolamarckia cadamba is classified under the Rubiaceae family. It is a type of fast-

growing deciduous tree species. N. cadamba have different common names in different

regions. For example, it is commonly called Kelampayan in Malaysia, burr-flower tree in

England, Bangkal in Brunei, and Jabon in Indonesia. This tree species usually has a

medium to large size, with maximum height of 45 meter and trunk diameter ranging from

100 cm to 160 cm. The tree trunk is straight and cylindrical with branches spreading

horizontally and arranged in tiers (Krisnawati et al., 2011). Kelampayan has a rounded,

umbrella-shaped crown. The leaves of N. cadamba are green and glossy, with oval or

elliptical shape. The flowers are often small, yellowish or orange coloured with dense

globose heads (Gautam et al., 2012). Kelampayan is native in South Asia and Southeast

Asia countries such as like Malaysia, Indonesia, Cambodia and India. Besides, it also

thrives in Australia, Papua New Guinea and China (Krisnawati et al., 2011).

With increasing urbanization and industrialization, Kelampayan is now emerging

as an important timber resource. N. cadamba has good economical prospects owing to its

rapid growth rate and self-pruning characteristic and high quality wood texture. It is a

lightweight hardwood with fine texture. N. cadamba is widely harvested for various

purposes, such as plywood production, pulping, raw materials for light constructions and

4

furniture manufacturing (Nair, 2007). Studies also revealed that N. cadamba has great

medical potential as it contains many phytochemicals like flavonoids, alkaloids, saponin

and terpenoids. In India, it is often used to treat fever and eye inflammation. The barks

and leaves of N. cadamba are reported to possess antioxidant activity, antimicrobial

activity, wound healing and antidiabetic effects (Bussa & Pinnapareddy, 2010; Gautam et

al., 2012).

(a) (b)

(c) Figure 2.1 N. cadamba. (a) The tree of N. cadamba, (b) the leaves of N. cadamba, and (c) the flower of N.

cadamba.

(Adapted from

(a) http://www.flickr.com/photos/61649195/6059370413/sizes/l/in/photostream/

(b) http://www.inmagine.com/dinodiarf-010/ptg01958655-photo

(c) http://www.pictureworldbd.com/Flower/kadam-flower-picture.htm)

5

2.2 Flavonoids in plants

Flavonoids are the largest subgroup of secondary metabolites that are found in various

plants (Tahara, 2007). Flavonoids comprise of numerous and diverse family of aromatic

molecules which are mainly derivatives of Phe and malonyl-coenzyme A. Flavonoids can

be divided into six major subgroups in higher plants, namely, chalcones, flavones,

flavonols, flavandiols, anthocyanins, and proanthocyanidins. With a wide range of

physical structures, flavonoids play various roles that are essential for the survival of

higher plants. It was reported that flavonoids are imperative in UV protection, pollen

fertility, auxin transport regulation and pigmentation (Winkel-Shirley, 2001). The

infections of pathogens like bacteria and fungi also trigger the production of flavonoids,

suggesting the function of flavonoids in combating pathogen attacks. Although the

biosynthesis of flavonoids can also be influenced developmental factors, the

environmental stressors such as wounds, strong lights, cold, and drought are the common

inducers of flavonoids synthesis (Guo et al., 2008).

Flavonoids biosynthesis involves the phenylpropanoid metabolic pathway. The

amino acid phenylalanine is the initial intermediate in this pathway (Shih et al., 2008).

During the flavonoids synthesis pathway, there are several branch points at chalcone

synthesis, leading to different end-products. The hydroxylation of flavanone by the

flavanone 3-hydroxylase is a key step for flavonols and anthocyanin biosynthesis (Kim,

et al., 2008).

6

Figure 2.2 The biosynthesis pathway of flavonoids. (Adapted from Albert et al., (2009). Light-induced

vegetative anthocyanin pigmentation in Petunia. Journal of Experimental Botany, 60 (7), 2191-2202)

2.3 Anthocyanin

One of the major end products catalysed by flavanone 3-hydroxylase is anthocyanin.

Anthocyanin is a water-soluble pigment that can be found abundant in the vacuoles of

vegetative tissues. Studies showed that anthocyanin plays a significant role in stress

tolerance and plant defense. Anthocyanin often associates with biotic and abiotic

stressors, indicating its ability in alleviating the stress impact on plants, which directly

correlates with the environmental fitness of plants.

7

One of the key functions of anthocyanin is photoprotection. According to Hatier

and Gould (2009), the light filtering ability of anthocyanin helps to shield the

photosynthetic cells from the adverse effect of excessive light flux. This prevents the

occurrence of photoinhibition, whereby the quantum yield of photosynthesis is decreased

due to excessive light. Moreover, there is also scientific evidence that the cyanic leaves,

which are rich in anthocyanin, have higher photosynthetic recovery than acyanic leaves.

According to Hughes et al. (2007), not only correlating with stress response, the

biosynthesis of anthocyanin also depends on the developmental stage of plant organs.

Anthocyanin is persistently produced in developing leaf until it is fully differentiated, and

subsequently declines when the sufficient photosynthesis rate can be achieved. This

proves the role of anthocyanin in photoprotection before the other photoprotection

mechanisms mature. Apart from that, anthocyanin is also crucial for UV protection. UV

rays are detrimental to the plants as it can damage the cells by generating photoproducts

in DNA and disrupt the protein structures in plant cells (Gerhardt et al., 1999; Sinha and

Häder, 2002). Previous studies showed that the accumulation of anthocyanin in leaves

can filter the UV-A and UV-B photons, and therefore protecting the plants from the

cellular damages caused by UV radiations (Guo et al., 2008).

Another primary role of anthocyanin is to protect the plants from free radical

scavenging through antioxidative activity. Environmental stressors like excessive light or

UV radiation can induce the free radicals formation in plant cells. These free radicals,

mostly comprises “reactive oxygen species” (ROS), contain one or more unpaired

8

electrons. Overabundance of ROS is detrimental to the plant as it can damage structure of

the phospholipid membranes, proteins and nucleis acids. Hence, anthocyanin acts as the

antioxidant to control the concentration of ROS (Hatier & Gould, 2009).

Apart from that, anthocyanin also functions to enhance the tolerance of plants

towards extreme temperature, drought stress and wounding. Besides, previous studies

also stated that anthocyanin is important in antipathogenic activities, osmoregulation and

heavy metal chelation, recruitment of pollinators and seed dispersers (Tereshchenko, et al,

2012 (Winkel-Shirley, 2001).

Figure 2.3 The general structure of anthocyanin. (Adapted from http://www.micro-

ox.com/chem_antho.htm)

9

2.4 Flavanone 3-hydroxylase

Flavanone 3-hydroxylase (EC 1.14.11.9) is one of the key enzymes involved in flavonoid

synthesis pathway (Kyoto Encyclopedia of Genes and Genomes; UniProt database). It is

also known as flavanone 3-dioxygenase, naringenin 3-hydroxylase, or flavanone synthase

I. Flavanone 3-hydroxylase is an oxidoreductase that catalyses the hydroxylation of α-

flavanone at the 3 position of the C-ring, resulting in the formation of dihydroflavonols,

which is the precursor of flavonols and anthocyanins, and catechins (Wellman et al.,

2004). Iron and ascorbate are the cofactors for flavanone 3-hydroxylase. The chemical

reaction of flavanone 3-hydroxylase (F3H) is shown below:

α -flavanone + 2-oxoglutarate + O2 = α-dihydroflavonol + succinate + CO2

2.5 Flavanone 3-hydroxylase gene

Flavanone 3-hydroxylase gene is one of the structural genes that are involved directly in

the biosynthesis of flavonoids. F3H gene has been isolated from a number of plants,

including maize, snapdragon, and petunia (Guo et al., 2008). From previous studies, the

amino acid sequence of F3H was confirmed to be highly conserved among plant species.

Scientific evidence had shown that F3H gene was expressed in pigmented tissues and

associated with the accumulation of its end-products like flavonols, anthocyanins, and

catechins (Deboo, et al., 1995).

10

In tea, flavanone 3-hydroxylase exhibits a positive relationship with the

concentration of catechins (Kim, et al., 2008). The positive correlation between the

concentration of catechins and CsF3H expression in leaves of different developmental

stages, suggesting that F3H gene is transcribed in different levels according to the

developmental stages of plants. CsF3H expression was also shown to be down-regulated

in response to drought, abscisic acid and gibberellic acid treatment, but up-regulated in

response to wounding. The strong correlation between the concentration of catechins and

CsF3H expression indicates a critical role of F3H in catechin biosynthesis (Singh et al.,

2008). According to Pelt, et al. (2003), the expression of flavanone 3-hydroxylase gene is

the key factor to determine the flavanone usage in plants, which in turn influence the

patterns of flavonoid accumulation.

11

CHAPTER 3

MATERIALS AND METHODS

3.1 Plant Material Sampling

The young leaves and developing xylem tissues were sampled from a Kelampayan tree

from the natural stand at Kota Samarahan area. The collected young leaf tissues were

then labeled and stored at -80°C prior to RNA extraction to prevent the wilting of leaves

and degradation of total RNA.

3.2 Total RNA Isolation

A small section of tissue from young leaves was collected from Kelampayan tree and its

total RNA was isolated by using RNeasy Midi Kit with modification (Qiagen, Germany).

The tissues were weighed and frozen by using liquid nitrogen. The frozen tissues were

then ground into fine powder by using mortar and pastel. After that, the ground powder

was transferred into a clean 15 ml centrifuge tube. 5.0 ml of Buffer RLT and 1% β-

mercaptoethanol was subsequently added into the tube. The mixture was vortexed gently

and centrifuged for 10 min at room temperature at 3,000 rpm. After that the supernatant

was transferred into a new 15 ml falcon tube. A total of 0.5 volume of 100% ethanol was

added and mixed by gently inverting the tube. The mixture was then transferred into the

RNeasy midi column and inserted into a 15 ml centrifuge tube. Subsequently, the mixture

was centrifuged for 5 min at 3,000 rpm. The centrifugation was repeated until all the

12

supernatant is eluted. A total of 2.5 ml of Buffer RWI was then transferred into the

RNeasy column and the mixture was centrifuged again for 5 min at 3,000 rpm at room

temperature. The flow through was discarded. A total of 20.0 ml DNase I incubation mix

solution was then added to the tube and left on bench for 15 minutes. The column was

then taken for centrifugation for 5 min at 3,000 rpm and the flow through was discarded

again. A total of 2.5 ml of Buffer RPE was transferred into the column and centrifugation

was performed for 5 min at 3,000 rpm.

Then, the column was taken out and placed into a new 15 ml falcon tube. Two

hundred microliters of DEPC treated water was subsequently pipetted into the centre of

column and left perpendicularly for 1 minute. Centrifugation was performed on the tube

for 3 min at 3,000 rpm. The eluted solution was then transferred to the first elution tube.

The elution step was repeated until the second elution was obtained. The isolated total

RNA was then checked through agarose gel electrophoresis using 1.0% (w/v) gel

concentration.

3.3 Agarose Gel Electrophoresis

The total RNA was analyzed through 1.0% (w/v) agarose gel electrophoresis using. A

total of 0.4 g agarose powder was weighed and mixed with 40ml of 1× TAE buffer in a

conical flask. The mixture was then heated in at 300°C for 2 minutes to ensure that the

gel was completely dissolved. The mixture was then left to cool down and poured into the

casing. The gel was left to further cooled down and solidify. Three microliters of RNA

solution was mixed with 1.0 µl of loading dye via repetitive pipetting and loaded into the

13

sample well of the agarose gel. The electrophoresis was started at 50 V, 40 A for 90

minutes. Later, the ethidium bromide was used to stain the gel for 10 seconds, followed

by de-staining with distilled water for 30 minutes. The RNA bands were then observed

by viewing under a UV transilluminator.

3.4 RNA Purity Determination and Quantification

A total of 3.0 ml of purified RNA was added into 2997.0 ml of ultra pure water in a quartz

cuvette and the light absorbance was measured using spectrophotometer. Absorbance

reading was recorded at wavelengths of 230 nm, 260 nm and 280 nm. The concentration

of isolated RNA can be estimated though the formula:

[RNA] (ng/ µl) = A260 × 40 µg/ ml

The absorbance ratio of A260/A280 and A260/A230 was be calculated to determine the purity

of the RNA.

3.5 Primer Designing

The data mining of the specific primer pair was performed by retrieving related

sequences from the NCBI GenBank database. The amino acid and nucleotide sequences

from other plant species was searched and retrieved from the NCBI Genbank database

14

(http://www.ncbi.nlm.nih.gov/nucleotide/). The nucleotide sequences of five woody plant

species were retrieved from the database as follows:

Pyrus pyrifolia (Accesion no. GU390545)

Malus domestica (Accesion no. AF117270)

Prunus persica (Accesion no. HM543570)

Litchi chinensis (Accesion no. HQ402912)

Juglans regia (Accesion no. FJ966204)

By using ClustalW software (http://www.ebi.ac.uk/Tools/clustalw2/index.html),

multiple alignments were performed on the sequences to examine for the conserved

regions among the plant species. Then, the forward and reverse primers were designed

based on the partial cDNA nucleotide sequence of Juglans regia (Accesion no. FJ966204)

by using the Primer Premier 6.0 software. The sequence of the forward primer was 5’

TTCATTGTCTCCAGCCATC 3’ while the sequence of the antisense primer was 5’

GGTCCTTGCTCATCTTCC 3’. The primer pairs generate an amplicon with ~652 bp

expected size.

3.6 RT-PCR and Optimization of Polymerase Chain Reaction (PCR)

The first strand of cDNA was synthesized from the isolated RNA according to the

protocols of Ready-to-go You-Prime First-Strand Beads (GE Healthcare, USA). The

synthesized cDNA was then used as the template for synthesizing double stranded cDNA