Molecular cloning and sequence variation of cDNA encoding for trypsin inhibitor from

Kelampayan (Neolamarckia cadamba)

Melanie Ann Perera

(21435)

Bachelor of Science with Honours

Resource Biotechnology

2011

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

Faculty of Resource Science and Technology

Molecular Cloning and Sequence Variations of cDNA Encoding for Trypsin Inhibitor

from Kelampayan (Neolamarckia cadamba)

Melanie Ann Perera

(21435)

Bachelor of Science with Honours

Resource Biotechnology

2011

I

ACKNOWLEDGEMENTS

The highest thanks goes to God for seeing me through the good and bad times of this

project. To my beloved Mum, Dad and sister, thank you for being my emotional and

spiritual support whenever I needed it. To my supervisor Dr. Ho Wei Seng and

co-supervisor, Dr. Pang Shek Ling, thank you for providing the information and training

required. I truly appreciate all that I have learned through my project in the Forest

Genomics and Informatics Laboratory (fGiL). A special thank you to Ms. Tchin Boon Ling

(Msc. Student) and Ms. Tiong Shing Yiing (Msc. Student) for your patience, guidance and

help. To all the other Msc. Students and my beloved lab-mates, I thank each one of you for

your support in various ways. A big thank you also to UNIMAS and the Faculty of

Resource Science and Technology (FRST) for providing the means for this project to be

carried out successfully. And last but not least, my lecturers, friends, lab assistants and

acquaintances thank you all very much for helping me in one way or another in

successfully completing my project.

II

DECLARATION

I hereby declare that this thesis is based on original work except for quotations and

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

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

__________________________________

MELANIE ANN PERERA, 21435

Date:

Resource Biotechnology

Department of Molecular Biology

Faculty of Resource Science and Biotechnology

Universiti Malaysia Sarawak (UNIMAS)

III

TABLE OF CONTENTS

ACKNOWLEDGMENT I

DECLARATION II

TABLE OF CONTENTS III

LIST OF ABBREVIATIONS VII

LIST OF TABLES IX

LIST OF FIGURES XII

ABSTRACT / ABSTRAK XIII

SECTION I INTRODUCTION 1

SECTION II LITERATURE REVIEW 7

2.1 Selection of species studied (Neolamarckia cadamba) 7

2.1.1 Family Rubiceae 7

2.1.2 Morphological characteristics 7

2.1.3 Economical Importance 9

2.2 Proteinase inhibitors (PIs) 10

2.3 Trypsin inhibitors 11

2.4 Miraculin protein 14

2.5 Miraculin-like proteins 15

2.6 Programmed plant cell death (PCD) 16

2.7 Single Nucleotide Polymorphism (SNP) 17

2.7.1 Applications of SNPs 18

IV

SECTION III MATERIALS AND METHODS 20

3.1 Plant samples 20

3.2 Total RNA isolation from Neolamarckia cadamba 22

3.3 Data mining for trypsin inhibitor sequence 23

3.4 Primer Design 24

3.5 PCR optimization 24

3.6 RT-PCR 25

3.7 PCR product purification 27

3.8 Molecular Cloning 28

3.8.1 Ligation 28

3.8.2 Transformation 29

3.8.3 Colony PCR 30

3.8.4 Plasmid isolation and purification 30

3.9 Sequence variation (Single nucleotide polymorphism) of

trypsin inhibitor in Kelampayan 32

3.9.1 Genomic DNA sample preparations 32

3.9.2 PCR product purification 33

3.10 Cloning of PCR fragments 33

3.10.1 Ligation 33

3.10.2 Transformation 34

3.11 Confirmation of the desired insert 35

3.11.1 Colony PCR 35

3.12 Plasmid isolation and purification 35

3.13 DNA sequencing and analysis 36

V

SECTION IV RESULTS AND DISCUSSION 37

4.1 RNA extraction and purification 37

4.2 Estimation of DNA concentration 38

4.3 Primer design 40

4.4 PCR optimization 40

4.5 PCR product purification 42

4.6 Cloning of PCR product 44

4.7 Confirmation of desired insert 47

4.7.1 Colony PCR 47

4.8 Plasmid purification 48

4.9 DNA sequencing and data analysis 50

4.9.1 Homology sequence of KTI gene 50

4.10 Sequence variations (Single nucleotide polymorphisms)

in trypsin inhibitor gene of Neolamarckia cadamba (Kelampayan) 51

4.10.1 DNA concentration optimization 51

4.10.2 Gel extraction and DNA purification 52

4.10.3 Cloning of PCR products 53

4.11 Confirmation of desired insert 54

4.11.1 Colony PCR 54

4.12 Plasmid purification 55

4.13 DNA sequencing and data analysis 57

4.13.1 Sequence variation in trypsin inhibitor gene

among the Neolamarckia cadamba trees 57

4.13.2 Identification of possible restriction enzymes for SNP sites 64

VI

SECTION V CONCLUSIONS AND RECOMMENDATIONS 69

REFERENCES 72

APPENDIX 82

VII

LIST OF ABBREVIATIONS

A Ampere

AGE Agarose gel-electrophoresis

β-ME Beta-mercaptoethanol

BLAST Basic Local Alignment Search Tool

b.p Base pairs

CAPS Cleaved-amplified polymorphic sequence

cDNA Complementary DNA

cm Centimeter

CTAB Cetyltrimethylammonium Bromide

ddH2O Double-distilled water

DEPC Diethylpyrocarbonate

dH2O Distilled water

DNA Deoxyribonucleic acid

dNTP deoxyribonucleotide triphosphate

GSP Gene-specific primer

IPTG Isopropyl β-D-1-thiogalactopyranoside

KTI Kunitz trypsin inhibitor

LB Luria Bertani/Broth

m Meters

μl Microliter

min Minute

mm Milimeter

NaOAc sodium acetate

NCBI National Centre for Biotechnology Information

PC Phenol-Chloroform

VIII

PCR Polymerase Chain Reaction

Proteinase inhibitors PIs

PVP polyvinylpyrrolidinone K30

QTL Quantitative trait locus

RACE Rapid Amplification of cDNA Ends

RNA Ribonucleic Acid

RNase Ribonuclease

rpm Revolution per minute

r.t. Room temperature

RT-PCR Reverse Transcription Polymerase Chain Reaction

sec Second

SSC Sodium chloride sodium citrate

STI Serine trypsin inhibitor

Ta Annealing temperature

Tm Melting temperature

TAE Tris-Acetate EDTA

V Volt

IX

LIST OF TABLES

Table Page

3.0 Leaf samples, tree measurements and location in trial plot 20

3.1 Characteristics of Kelampayan trees from which samples were taken 21

3.3 Mastercycler Gradient PCR reaction mixture, concentration, and volume 25

3.4 Components for first-strand cDNA synthesis 26

3.5 PCR mix 26

3.6 Ligation reaction mixture and volume 28

3.7 Colony PCR reaction mixture, concentration and volume 30

3.8 PCR reaction mixture, concentration and volume 33

3.9 Colony PCR profile 35

4.0 Estimated RNA concentration and RNA purity 39

4.1 Column number and annealing temperatures (°C) 41

4.2 Optimized thermal cycling profile 42

4.3 Concentration of Lambda HindIII DNA ladder bands 43

4.4 BLASTn output for amplified partial KTI gene 51

4.5 Blue-white screening results on six LAIX plates 53

4.6 BLASTn output for amplified partial trypsin inhibitor DNA 57

4.7 BLASTp output for amplified partial trypsin inhibitor DNA 58

4.8 SNPs detected in samples B, M, and N 59

X

4.9 Possible restriction enzymes and their cut site numbers for sample B 66

4.10 Possible restriction enzymes and their cut site numbers for sample M 67

4.11 Possible restriction enzymes and their cut site numbers for sample N 68

XI

LIST OF FIGURES

Figure Page

1.0 (a) Ribbon structure of soybean Kunitz type trypsin inhibitor (STI) 4

(b) STI Surface structure 4

1.1 Ribbon structure of miraculin-like protein with the 12 β-sheet structure

of the Kunitz STI protein. The sheets are classified into three groups-

Blue sheets A1 to A4, green sheets B1 to B4, and orange sheets C1 to C4 5

2.1 Neolamarckia cadamba

(a) Lepidopteran Stem borer Endoclita aroura 8

(b) Flowers and leaves 8

2.2 Red berries (miracle fruit) of the Richadella dulcifica shrub 14

4.1 5S rRNA, 18S rRNA and 25S rRNA bands from isolated total RNA on a 1.5%

(w/v) agarose gel. Lanes D1 and D2: Elute 1 and 2 of total RNA isolated 38

4.2 Single banding from Ta optimization of RNA sample 42

4.3 Gel electrophoresis of purified PCR product on a 1.5% agarose gel 44

4.4 Blue-white screening on two LAIX plates

(a) C1, C2, and C3 marked 45

(b) C4, C5, and C6 marked 45

4.5 Gel electrophoresis of colony PCR result on a 1% (w/v) agarose gel 48

4.6 Gel electrophoresis of purified plasmid on a 1.0% agarose gel 49

4.7 Gel electrophoresis of DNA samples with different concentrations

XII

on 2% agarose gel 52

4.8 Gel electrophoresis of purified genomic DNA samples from Kelampayan

trees on 1.5% (w/v) agarose gel 53

4.9 Gel electrophoresis showing colony PCR results on 1.5% (w/v) agarose gel 55

4.10 Gel electrophoresis of purified plasmids 56

4.11 Alignment of partial trypsin inhibitor gene from six Kelampayan trees

(A, B, P, M, N, and W) using CLC Free Workbench 4 61

4.12 Alignment of translated nucleotide sequences

(Samples A, B, P, M, N and W) 64

4.13 (a), (b) and (c) Positions of restriction enzymes for SNPs on sample B 65

4.14 (a), (b) and (c) Positions of restriction enzymes for SNPs on sample M 67

4.15 (a), (b) and (c) Positions of restriction enzymes for SNPs on sample N 68

XIII

MOLECULAR CLONING OF cDNA ENCODING TRYPSIN INHIBITOR FROM KELAMPAYAN

(Neolamarckia cadamba)

MELANIE ANN PERERA

Resource Biotechnology

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

ABSTRACT

Proteinase inhibitors are important in plant defense mechanisms. The gene that is responsible for it is

Kunitz-type trypsin inhibitor (KTI) which reduces the digestability and nutritional quality of the leaves of the

tree which is infected by insect pests or microbes. The objectives of this study are to clone the cDNA

encoding for trypsin inhibitor and to identify single nucleotide polymorphisms (SNPs) through in-silico

analyses. Neolamarckia cadamba (Kelampayan) is used in this study because of its fast growth rate and

economic importance. Total RNA was isolated from developing xylem samples and reverse transcribed into

cDNA. The ~323 bp gene was then amplified and cloned into pGEM-ⓇT Easy vector systems and sent for

automated sequencing. Results showed that a miraculin-like gene was isolated. Further analysis revealed that

the gene belongs to the plant Kunitz serine trypsin inhibitor (STI) family of proteinase inhibitors, with the

same functions as KTI gene. A total of 27 SNPs were identified with 22 synonymous and 5 non-synonymous.

Keywords: cDNA, Neolamarckia cadamba (Kelampayan), trypsin inhibitor, miraculin-like gene, and single

nucleotide polymorphism (SNP)

ABSTRAK

Gen dalam system pertahanan pokok ialah sejenis proteinase inhibitor, Kunitz-type trypsin inhibitor (KTI)

yang mengurangkan kadar nutrisi daun serta mengganggu penghadaman serangga perosak. Objektif kajian

ini ialah mengklon cDNA yang menyandi gen trypsin inhibitor dan mengenalpasti single nucleotide

polymorphisms (SNPs) melalui analisis in-silico. Neolamarckia cadamba (Kelampayan) diguna dalam kajian

ini kerana kadar pertumbuhan yang pantas dan kepentingan ekonominya. RNA keseluruhan diasingkan dari

tisu xylem yang sedang tumbuh dan ditranskripsi kepada cDNA. Gen ~323 bp itu diamplifikasi, diklon ke

dalam sistem vektor pGEM-ⓇT Easy dan dihantar untuk sequencing. Gen miraculin-like telah diasingkan.

Kajian mendapati kedua-dua trypsin inhibitor dan miraculin-like adalah dari keluarga gen yang sama

dengan fungsi yang sama. 22 SNPs synonymous dan 5 non-synonymous dijumpai.

Kata kunci: cDNA, Neolamarckia cadamba (Kelampayan), trypsin inhibitor, gen miraculin-like, dan single

nucleotide polymorphisms (SNPs)

1

SECTION I

INTRODUCTION

Forest tree molecular biology enables a better understanding of the many natural

populations of woody trees besides providing ways to genetically improve commercially

useful plant sources for fuel, fibre, timber and other industries (Bradshaw et al., 1989).

Increasing demand for tropical timber and the shortage of more popular

commercial timbers (Chong, 1979) such as Rubberwood (Hevea brasiliensis) and their

high prices has made wider use of the lesser known commercial timbers such as

Kelampayan (Neolamarckia cadamba) as timber stock (Lim et al., 2005). N. cadamba is

the botanical name of a fast-growing tree species (Ismail et al., 1955) of the family

Rubiaceae. They can grow up to 45 m in height, without branches for more than 25 m with

a diameter of 100-160 cm. (Lim et al., 2005; Peter, 2007). The flowers are globose and

solitary, orange or yellow in colour (Acharyya et al., 2010) and the fruits are small

capsules of dense fleshy, yellow or orange infructescence with approximately 8,000 seeds.

The fruits have medicinal values in curing ulcers, diarrhea, fever, and vomiting (Peter,

2007). Trees are well distributed from India to Papua New Guinea. It is a favoured species

in reforestation projects as they can grow very well in bare and exploited lands (Ismail et

al., 1955). Lim et al. (2005) reported that these trees are distributed in lowland to mountain

2

forests (1000 m altitude); growing in deep, moist alluvial soil (Choo et al., 1999), often by

streams and rivers (Nair, 2007).

Large scale forest plantations have undeniably contributed to the occurrence of

disease outbreak and pest infection (Farid et al., 2008). According to Balestrazzi et al.,

2006, a considerable number of damages in woody plant species are caused by insects,

resulting in severe reduction of growth rates and production (about one-third of the total

potential pre-harvest production).

The major defoliator of N. cadamba is the caterpillar Artroschita hilaralis (Nair,

2007). Woody plant components generally make pest control more costly (devising new

control strategies, improving on the previous methods, obtaining new pesticide and

equipment) as the trees tend to build up their insect pest problems as years go by (Epila,

1986). Intense studies on various pest and diseases reveal that injuries or attack by insects

or pathogens bring about the expression of proteinase inhibitors, which are important plant

proteins for plant defense against pest and in the proteolysis regulation in plant

development (Mondego et al., 2010). These chemical defenses are often effective against a

wide range of organisms, thus hampering attacks through recognition mechanisms that

identify the organism and activate specific defenses against it (Franceschi et al., 2005).

3

Trypsin inhibitors (Figure 1.0) are a type of serine proteinase inhibitor which is

produced in response to insect feeding (Mondego et al., 2005) and are found in storage

tissues in plants (Pandey and Jamal, 2010; Chen and Mitchell, 1972; Major and Constabel,

2008). They are divided into two families, Kunitz type (approximately 20-kDa with one or

two disulphide bonds and one reactive site) and Bowman-Birk type (approximately 8 to

10-kDa with seven disulphide bridges, a high Cys content and two reactive sites). These

proteinase inhibitors inhibit serine proteases, in particular trypsin (Major and Constabel,

2008). They act as competitive inhibitors by binding tightly to the active sites of trypsin,

the protease (Beynon and Bond, 1989). Trypsin inhibitors act as inhibitors of insect-gut

proteinases (Bradshaw et al., 1989) and reduce the digestability and nutritional quality of

the leaves of the tree which is infected by insect pests. Recent studies in poplar trees

showed that their defense responses against insects displayed very prominent Kunitz

protease inhibitors (KPIs) (Philippe et al., 2009). Therefore with the high throughput

validation and transgenic techniques which are available at present times, crop plants with

enhanced resistance can be produced in the agricultural sector (Chen, 2008).

4

(a) (b)

Figure 1.0 (a) Ribbon structure of soybean Kunitz type trypsin inhibitor (STI). Green arrows, a red ribbon

and light pink ropes represent β-strands, a 310-helix and the loops, respectively. (b) STI Surface structure.

Positively charged regions are blue and negatively charged regions are red. (Adapted from Song and Suh,

1998)

Apart from trypsin inhibitors, miraculin-like proteins (MLPs) (Figure 1.1) are also

a part of the plant Kunitz serine trypsin inhibitor (STI) family of proteinase inhibitors (PIs)

with three disulphide bridges as compared to two conserved disulphide bridges in trypsin

inhibitors (Mondego et al., 2010). These proteins show sequence similarities to miraculin

(a taste modifying protein) hence the name.

5

Figure 1.1 Ribbon structure of miraculin-like protein with the 12 β-sheet structure of the Kunitz STI protein.

The sheets are classified into three groups- Blue sheets A1 to A4, green sheets B1 to B4, and orange sheets

C1 to C4 (Adapted from Mondego et al., 2010)

These genes that have been identified can further be analyzed for single nucleotide

polymorphisms (SNPs). SNPs are genomic sequences of an organism which has a single

nucleotide difference between populations of the same species (Gupta et al., 2001). These

sequence differences can be used as genetic markers for plant improvement programs (Nur

Fariza et al., 2008) especially in improving plant defense mechanisms.

To date, little is known of trypsin inhibitors or miraculin-like proteins, especially in

forest tree species. The concerns weighing on good quality wood for timber industry to

meet the increasing demand has made efforts for plant defense mechanism discoveries and

improvements very much needed. Therefore the objectives of this study would be to obtain

the partial cDNA of trypsin inhibitor from Neolamarckia cadamba, to perform in-silico

6

analysis of trypsin inhibitor gene based on the partial cDNA sequence generated in the

present study and to identify the single nucleotide polymorphisms in the sequences.

7

SECTION II

LITERATURE REVIEW

2.1 Selection of species studied (Neolamarckia cadamba)

2.1.1 Family Rubiaceae

The family Rubiaceae consists of mainly tropical woody plants and is monophyletic

(Mongrand et al., 2004). It is one of the largest of angiosperm families consisting of three

subfamilies and close to 10,000 species. The three subspecies are Rubioideae,

Cinchonoideae, and Ixoroideae. The subspecies Rubioideae are identified by a combination

of morphological characteristics such as having valvate aestivation, herbaceous habit and

articulate hairs. Cinchonoideae members are woody, and have imbricate aestivation while

Ixoroideae has contorted aestivation and stylar pollen (Bremer et al., 1999).

2.1.2 Morphological characteristics

Neolamarckia cadamba (Roxb.) Bosser (Kelampayan) trees are large, deciduous and fast

growing. They are well distributed from India to Papua New Guinea at 650 to 1000m in

altitude (Ismail, 1955) and can grow up to 45 m tall with bole diameter of 100 to 160 cm.

Their flowers are yellow to orange in colour, usually globose heads, about 55 mm in

diameter. The fruit of the tree is in small capsules, about 45 mm in diameter, closely

8

packed to form fleshy orange or yellow infructescence covered with short bristles with

about 8,000 seeds. Their branching is tiered with simple leaves of about 13 to 32 cm long

(Joker, 2000). Its timber is light, with a moderately coarse and even texture (Lim et al.,

2005). They self-prune and grow well in bare and exploited lands (Ismail, 1955).

The caterpillar Artroschita hilaralis has been found to be a major defoliator of N.

cadamba. Others include the hornworm, Daphnis hypothous and the hepialid caterpillar

Sahyadrassus malabaricus (Nair, 2007) and lepidopteran stem borers (Endoclita aroura).

(a) (b)

Figure 2.1 Neolamarckia cadamba. (a) Lepidopteran Stem borer Endoclita aroura and (b) Flower and leaves

[Adapted from (a) http://www.jircas.affrc.go.jp/english/publication/jarq/37-4/37-04-08.pdf and (b)

http://www.istockphoto.com/stock-photo-10789657-the-kadamba-flower.php

9

2.1.3 Economical importance

Malaysia’s main markets for timber are Japan, China, India, the Middle East, USA and

Europe (Noma, 2009) In Malaysia, Kelampayan was grown in plantations as early as 1953

and 1961 at Sibunga Forest Reserve near Sandakan, Sabah (Ismail et al., 1995). Forest

plantations already began to grow rapidly since 1980s in Peninsula Malaysia due to the

forecasted shortage of timber from natural forests (Chong, 1979 cited in Farid et al.,

2008). It dominates the initial re-growth stages of tropical secondary forests, especially on

moist sites (Ismail et al., 1995). Today, its timber is used for plywood manufacture,

packing cases, wooden sandals, toys, disposable chopsticks (Choo et al., 1999), picture

frames, pulp to produce low and medium quality paper (Joker, 2000), moulding and

skirting as the timber is soft and light (Lim et al., 2005). The wood can also serve in light

construction work (indoors only) because it is perishable when in contact with the ground

(Joker, 2000). Medical use of this tree is reported to be as an astringent anti-hepatotoxic

(Patel and Kumar, 2010) and an effective treatment for diabetes mellitus, venereal disease

and peptic ulcers (Bussa and Pinnapareddy, 2010).