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This is for Institutional undertaking

This is for Your undertaking

Acknowledgement
The six year course in integrated M. Tech (Biotechnology) covers the major area of Biological
Sciences and Technology. The project has been a major impetus in understanding the difference
between theoretical knowledge and its practical application.
I would like to thank my Sir Dr. M. Manickavasagam Department of Biotechnology and
Genetic Engineering supports my project.
I would like to wish my sincere thanks to the Head of Molecular Biology Division, and
Bhabha Atomic Research Centre. I express my heartful thanks to my project guide Dr. Ajay
Saini, MMGE Section, Molecular Biology Division, BARC for assigning me this project and his
guidance.
I would like to wish my sincere thanks to Mr. Ravi Prakash Sanyal for his suggestions
and support at every step during the progress of the work.
Finally special thanks to my parents and my friends for help and encouragement during
my project.

J.E. Mahendra Gupta

INDEX
Abbreviations

3-4

List of tables

List of figures

1. Introduction

7-10

2. Materials and Methods

11-28

2.1 Plant growth condition

11

2.2 Agarose Gel Electrophoresis

12

2.3 Staining of Agarose Gel

13

2.4 RNA Extraction

13

2.5 Quantification of RNA

14

2.6 cDNA synthesis

15

2.7 PCR Amplification of Cu/Zn SOD

16

2.8

Purification of PCR products

17

2.9

Restriction Digestion confirmation

18

2.10 Restriction digestion for cloning

19

2.11 Ligation

20

2.12 Purification of ligation product

20

2.13 Preparation of bacterial stock

20
1

2.14 Preparation of electrocompetent cells

21

2.15 Transformation

21

2.16 Screening of transformants

22

2.17 Plasmid isolation

23

2.18 Preparation of chemical competent E. coli cells

24

2.19 Protein overexpression

25

2.20 SDS- PAGE

26

3. Results

29-37

4. Discussion

38-41

5. Reference

42-46

Abbreviations
APS

Ammonium persulphate

bp

Base pair

cDNA

Complementary Deoxyribo Nucleic Acid

DNA

Deoxyribo Nucleic Acid

DNase

Deoxyribonuclease

dNTPs

Deoxy Nucleoside Tri Phosphates

DEPC

Diethylpyrocorbonate

EDTA

Ethylene Diamine Tetra Acetic Acid

FP

Forward primer

gm

Gram

hr

Hour

IPTG

Isopropyl -D-1-thiogalactopyranoside

Kan

Kanamycin

kV

Kilo Volt

Molar

mg

Milligram

min

Minute

ml

Milli litre

mM

Milli molar
3

NEB

New England Biolabs

PCR

Polymerase Chain Reaction

RNase

Ribonuclease

RT

Reverse transcriptase

RP

Reverse primer

rpm

Revolutions per minute

SDS

Sodium Dodecyl Sulphate

TBE

Tris Borate EDTA

TEMED

N, N, N, N Tetramethylethylenediamine

Units

UV

Ultra violet

Microgram

Microlitre

Volt

List of tables

Page No

Table 1

Composition of Hoaglands No.2 Basal Salt Mixture

Table 2

Composition of 5X TBE buffer

13

Table 3

Growth of rice seedlings in control and stress

30

Table 4

RNA quantification

32

12

List of Figures

Page No

Figure 1

Effect of salinity on rice growth

30

Figure 2

Total RNA extracted from rice seedlings

31

Figure 3

PCR amplification of rice Cu/Zn SOD (Os03g22810)

33

Figure 4

Restriction digestion analysis cDNA

34

Figure 5

Map of pET28a (+) plasmid

35

Figure 6

DH5 E. coli pET28a Os03g22810 transformants

35

Figure 7

Colony PCR of the transformants

36

Figure 8

Plasmid DNA isolation

37

Figure 9

Overexpression of rice Cu/Zn SOD Os03g22810

38

Introduction:
1. Abiotic stresses
Abiotic components (temperature, light, water, macro- and micronutrients etc.) of the
environment are essential for all life forms and any deviation (lower/higher) in their optimum
levels often leads to abiotic stress to the organisms. Abiotic stress conditions are detrimental to
all to all organisms including plants, and are collectively major cause of reduced crop
productivity (Boyer, 1982; Mahajan and Tuteja, 2005). Plants being sessile are more prone to
abiotic stresses and have evolved diverse array of mechanisms to adapt or to contain stress
induced damage (Hirayama and Shinozaki, 2010). Stress responses at different levels are very
precisely regulated, well coordinated at different levels (transcription, post-transcription,
translation and post-translation etc.) and involve different categories of genes (Kreps et al., 2002;
Buchanan et al., 2005) and often show a complex cross talk among different pathways
(Shinozaki and Yamaguchi-Shinozaki, 2000; Zhu, 2002).
1.1. Impact of salinity on agriculture
Among different abiotic stresses, salinity is an important agricultural problem, as it affects more
than 6% total and ~20% of irrigated land globally (Tuteja, 2007). Furthermore, the agriculture
area affected by salinity is continuously increasing due to both natural factors as well as human
activities. Soil salinity results in increased accumulation of salt in leaves that causes premature
senescence, reduces supply of assimilates to the growing regions, resulting in decreased plant
growth (Munns et al, 1985). As salinity significantly reduces productivity of all crops,
development/ identification of salt tolerant varieties using classical and/or biotechnological
approaches is highly desirable and an active area of research (Vinocur and Altman, 2005; Roy et
al., 2011). However, as salt tolerance is a polygenic trait affected by several factors (Shannon
1984), a thorough understanding of different mechanisms involved is both important and
required to realize the above-mentioned goals (Chinnusamy et al., 2004; Sreenivasulu et al.
2007).
1.2.Effect of salt stress on crops
One of the important challenges in agriculture to keep up the crop productivity, and hence plant
breeders rely on the elite and stress tolerant genotypes available in a particular crop for use in
breeding programs. Different plant species show different levels of tolerance to salinity and
7

among cereals rice, a globally an important crop (Khush, 2005), is relatively more salt sensitive
compared to wheat, barley, alfalfa etc. (Munns and Tester, 2008). Considerable intra-species
variability in salt tolerance has also been reported in different crops e.g., wheat, barley and
sorghum (Munns and Tester, 2008), which is often employed in breeding programs to enhance
the tolerance of the cultivars.
Rice is also a salt sensitive crop, however considerable intra-species variability in salt tolerance
has been observed among various genotypes (Yeo and Flowers, 1982; Walia et al., 2005; Negro
et al., 2011). Rice plants also show different salt tolerance at different stages, with germination
and active tillering stages being more tolerant than panicle initiation, fertilization and early
seedling stages (Walia et al., 2005). Several previous studies have shown that salt tolerance is
due to additive effect of multiple loci/genes that are actively involved in effective stress
perception and adaptive response (Gregorio and Senadheera, 1993; Sreenivasulu et al. 2007).
1.3. Salt stress induced damage and adaptive molecular responses
The overall effect of stress conditions on plants is the outcome of several direct or indirect
components that affect in different ways. The plant deals with effects of primary stress (salinity)
as well as secondary stresses such as osmotic stress (results in reduced water availability), ion
toxicity (due to increased levels of Na+ and K+ inside the cell), oxidative stress (due to increased
ROS levels) (Munns and Tester, 2008). Therefore, the detrimental effects of salinity on plants are
not only due to ion toxicity, but also involve cellular damage due to osmotic imbalance, and
oxidative stress induced damage (Zhu, 2001; Munns and Tester, 2008). Consequently, the
complex salt stress response involves diverse mechanisms that allow plants to cope up with salt
stress: 1) mechanisms to maintain low cellular Na + levels by ion exclusion/compartmentation
(Serrano and Rodriguez-Navarro, 2001), 2) mechanisms to maintain the osmotic potential
accumulation of compatible osmolytes (Hasegawa et al. 2000), and 3) mechanisms to involving
antioxidant metabolites and antioxidant enzymes to reduce elevated levels of reactive oxygen
species (ROS) of oxidative stress (Gill and Tuteja, 2010). All of these molecular mechanisms
are aimed towards protection against the salinity induced cellular damage (Munns and Tester,
2008).
Salinity induced ion-toxicity is due to competition of elevated Na+ with K+, which is involved in
several biochemical reactions. Several mechanisms are involved in sensing, exclusion, and/or
compartmentalization of the Na+ to achieve low Na+/K+ ratio, resulting in detrimental effects due
8

to ion toxicity. Some important candidates involved are ion antiporters/ symporters such as 1)
High affinity K+ transporters (Horie et al, 2010), 2) salt overlay sensitive (SOS) pathway genes
(Ren et al. 2005), and 3) NHX family of proteins involved in cation/proton exchange (Hunte et
al. 2005). Maintenance of high cytosolic K +/Na+ ratio especially in shoots have been strongly
suggested to be crucial for salt tolerance of plants (Hauser and Horie 2010).
Salinity also affects the osmotic potential within the plant cells. In response, osmolytes or
compatible solutes (proline, betaine, trehalose, polyols etc.) are accumulated to maintain
osmotic balance (Hasegawa et al. 2000). These compatible solutes alleviate the affects of the
associated osmotic stress by primarily increasing the osmotic potential.
The stress conditions generally results in accumulation of reactive oxygen species (ROS) such as
superoxide radical, hydrogen peroxide, and hydroxyl radicals that are highly reactive and can
cause damage to cellular components due to oxidative stress (Karpinski et al., 1999; Foreman et
al., 2003). Several intrinsic antioxidant systems that include enzymatic scavengers (superoxide
dismutases, peroxidases, and catalases) and non-enzymatic antioxidants (ascorbate and
glutathione) are involved in maintenance of an intricate balance of ROS and prevents their over
accumulation (Mittler, 2002, Scandalios, 2005).
1.4. Role of antioxidant enzymes
Among antioxidant enzymes superoxide dismutases (SODs) are ubiquitous metalloenzymes that
comprise an important component of enzymatic mechanisms for scavenging excess superoxide
radical (O2) generated as a result of environmental conditions such as, abiotic and/biotic stress
(Alscher et al., 2002). Superoxide radical reacts with hydrogen peroxide to produce hydroxyl
radical (OH), a highly reactive free radical in the biological systems that damages membrane
lipids, nucleic acids and proteins (Bowler et al., 1992). Superoxide dismutases (SODs) converts
superoxide radical (O2 ) into hydrogen peroxide (H2O2) and Oxygen (O2) and thus protects cells
against damage due to oxidative stress (Fink and Scandalios, 2002). Based on metal cofactors
involved the SODs are classified into three groups viz. Cu-Zn SODs, Fe SOD and Mn SODs.
Their numbers and cellular localization is also different namely Cu-Zn SODs are localized in
chloroplast as well as cytosol (del Rio et al., 2002), Fe SOD in mitochondria and Mn SODs in
peroxisomes (Rio et al., 2003). The effects of salt stress on the antioxidant responses have been
studied in a number of plant species including wheat, rice, sorghum and pea (Dionisio-Sese and
Tobita, 1998; Hernandez et. al. 2000; Sairam et. al. 2005; Henrique et. al., 2005).
9

Rice genome contains seven loci that codes for different types of SODs, four of them codes for
Cu/Zn SODs, two codes for Fe SODs and one code for Mn SOD.
Objective:
The objective of the present study was to isolate full length cDNA, clone and express a cytosolic
Cu/Zn superoxide dismutase (Cu/Zn SOD, encoded by locus LOC_Os03g22810 from rice
(Oryza sativa)

10

2. Materials and Methods

2.1. Plant growth condition
Requirements:

Plant Material: Rice genotype NSICRc106 (IRRI, Manila).

Growth Media: 1.63gm/litre of Hoaglands No.2 Basal salt Mixture. (1X)

Stress Solution: 1X Hoaglands No.2 Basal salt Mixture + 150 mM Sodium Chloride.
Table 1. Composition of Hoaglands No.2 basal Salt Mixture
Ingredients
Potassium Nitrate
Calcium Nitrate
Magnesium sulphate
Ammonium phosphate monobasic
Magnesium choloride.4H20
Boric acid
Molybdenum trioxide
Zinc sulphate.7H20
Copper sulphate.5H20
Ferric tartrate
Total gm/litre

Milligrams/litre
606.60
656.40
240.76
115.03

1.81
2.82
0.016
0.22
0.08
5.00
1.63

Rice genotype NSICRc106 seeds were washed with sterile distilled water and then were surface
sterilized with 0.1% mercuric chloride (HgCl2) for 10 min. The seeds were washed 5 times with
sterile distilled water to remove the HgCl2 and kept for germination. Germinated seeds were
grown in Hoagland basal media and transferred in plant growth chamber for next 6 days. Plant
growth condition 28 C/26 C (day/night) and humidity 65 % was used. For stress treatment, six
day old rice seedlings were transferred to Hoagland basal media supplemented with 150mM
sodium chloride.

2.2. Agarose gel electrophoresis

(i) Agarose

11

2 % agarose gel prepared from routine agarose powder of sigma Aldrich (USA) by dissolving
in 1X TBE.
(ii) Electrophoresis buffer
A stock solution of 5X TBE buffer was prepared as follows:
Table 2 Composition of 5X TBE buffer (1 litre)
Components

Amount

Tris base

54.0 gm

Boric acid

27.5 gm

0.5 M EDTA(pH 8.0)

20 ml

A working stock of 1X was prepared by diluting the autoclaved 5X TBE (1:5) with distilled
water.
(iii) DNA sample loading Dye
100 ml of dye was prepared as follows:
0.25% bromophenol blue.
0.25% xylene cyanol FF.
30% glycerol.
Volume made up to 100 ml. It was mixed with PCR products in and loaded in the gel.
(iv) Electrophoresis apparatus
Electrophoresis was carried out in a horizontal submarine gel electrophoresis tank.

2.3. Staining of agarose gel

Ethidium bromide stain

12

Stock solution of 10mg/ml of ethidium bromide in distilled water was prepared and stored at
room temperature in dark bottles or bottles warp with aluminum foil, working solution of
0.5 g/ml was prepared in 1X TBE.

2.4. RNA extraction

2.4.1. Isolation of tissue:
The shoot height was measured from day-6 (0-day time-point for stress treatment) to day-11 (5day time point after stress treatement) and at respective time points shoot samples were isolated
from control and stress seedlings, immediately frozen in liquid nitrogen and stored in -70 C till
further use.

2.4.2. Total RNA Extraction:

Total RNA was extract from shoot tissue of NSICRc106 by Trizol Reagent.

Requirements:

Homogenizer(Qiagen)

Liquid nitrogen

Trizol solution

Chloroform

Isopropanol

70% alcohol

DEPC water

DNase RNase free (Roche)

Protocol
13

Homogenization: 100 mg of tissue was homogenized in liquid nitrogen. 1ml of Trizol

Reagent was added to homogenized tissue and kept for 5 minutes at room temperature.
Phase separation: 400 l of chloroform was added and mixed well. Tissue sample was kept
in room temperature for 15 minutes and centrifuge at 10,000 rpm for 20 minutes at 4C.

Centrifugation to separate the organic phase and aqueous phase.

Precipitation: Aqueous phase was transfer into the new tube. 1 ml of isopropanol was added
to precipitate RNA and store in -20C for 30 minutes. Centrifuge at 13,000 rpm for 40

minutes at 4C and pellet was settled down.

Washing: The supernatant was removed and pellet was washed in 70% alcohol. Centrifuge

at 14,000 rpm for 10 minutes at 4C.

Solubilization: The pellet was allowed air dry and dissolved in 150 l of DEPC water.

Dissolved RNA incubated in 45-55C for 30 minutes and stored in -70C.

DNAse treatment: 1 l of DNAse added to remove any DNA present in RNA for 1 h at

37C followed by heat inactivation at 70C for 30 minutes.

Quality assurance: Integrity of the isolated RNA was analyzed on 1% agarose gel and gel
image was documented on gel documentation system (Syngene).

2.5. Quantification of RNA

Requirements:

Quartz cuvette (Hellma)

Total RNA
DEPC water
UV spectrophotometer (UV-1088)

Quantification of RNA was done by UV spectrophotometer. 5 l of RNA sample was mixed with
995 l of DEPC water and absorbance was measured at 260 nm. (1ml DEPC water was used as
blank). Dilution factor was incorporated in calculation to estimate quanitity.

2.6. cDNA synthesis

10 g of DNase treated was used for cDNA synthesis using anchored oligo dT 23 primer and
SuperScript II reverse transcriptase (RT).
14

Step 1: Denaturation:
Mater mix I:
RNA - 10 g
10 mM dNTPs -

1 l

Oligo dT23 (500 ng/ l) -

1 l

Reagents were mixed properly and incubated at 70C for 5 min. ( to denature secondary

structure)
After incubation tubes were quick chill in ice and centrifuged.

Master mix II (Enzyme mix preparation):

5X First strand synthesis buffer - 4 l
0.1 M DTT - 2 l
SuperScript RT II (200 U/ l) - 1 l
Reagents were properly mixed.

The master mix II was added into RNA containing tubes (master mix I), mixed and

centrifuged
Total reaction volume was 20 l / tube.

Reverse transcription programme:

Annealing: 25C for 10 min.
Extension:

42C for 2 h

Inactivation: 70C for 15 min

Stored at 4C
cDNA quantification:

cDNA was quantified by 1 l of cDNA sample and 999 l of distilled water in 1 ml quartz

cuvette.
Absorbance of the samples was measured at OD260.
15

2.7. PCR Amplification of Cu/Zn SOD:

a) In silico restriction analysis:

Bioinformatics tool NEB cutter was used for restriction enzymes analysis of Os03g22810.
Restriction enzymes present in multiple cloning sites (MCS) of pET28a plasmid was

screened for selection of restriction enzyme.

Based on the restriction map analysis NdeI and EcoRI enzyme sites were selected for cloning
into pET28a.

b) Primer designing:

Gene specific primers were designed for full length amplification of Os03g22810.
22-25 nucleotides sequence from 5 end of the cDNA was selected and NdeI restriction

enzyme site was incorporated towards the 5end of the primer.

Likewise, 22-25 nucleotides sequence from 3end of the cDNA was selected, reverse
complement the sequence and EcoR I restriction enzyme site was incorporated at the 5end of
the primer.

c) PCR amplification:

Full length Os03g22810 cDNA was PCR amplified from rice genotype NSICRc 106 using

Pwo DNA polymerase and gene specific primers.

All the PCR reagents were ice thawed and mixed.

Master mix preparation:

10 X buffer

2.5 l

10 mM dNTPs

1.0 l

10 mM MgCl2

1.0 l

Forward primer (10 pmole/ l)

0.5 l

Reverse primer (10 pmole/ l)

0.5 l
16

Taq DNA polymerase

0.2 l

Water

19.5 l

Total

25.0 l

Mastermix was mixed properly and aliquoted

PCR programme:
Initial denaturation: 94C - 10 min

Denaturation:

94C - 45 sec

Annealing:

60C - 45 sec

Extension:

72C - 1min 20 sec

Final extension:

72C - 10 min

Stored at

4C

35 cycles

3 l PCR product was loaded on 2.5 % agarose gel and electrophoresed.

Gel was stained with ethidium bromide solution.
Gel image was documented.

2.8. Purification of PCR products

Requirements:
Roche Purification Kit was used for PCR product purification.

High pure spin filter tubes.

High pure collection tubes.
Binding buffer.
Washing buffer.
Elution buffer.

Protocol

500 l of binding buffer was mixed with 100 l of PCR product in a tube.
Binding: A column was placed in a collection tube and solution was added to the column and

centrifuged for 1 minute at 13,000 g and flow through was discarded.

Washing:
i) 500 l of washing buffer was added and centrifuged for 13,000 g for 1 minute and flow
through was discarded.
ii) 200 l of washing buffer, centrifuged at 13,000 g for 1 minute was added.
17

Elution:
i) Column was transferred to a fresh collection tube.
ii) 50 l of elution buffer was added and centrifuged at 13,000 x g for 1 min and repeated
twice.

2.9. Restriction Digestion confirmation of Os03g22810 cDNA

Requirements:

10X cut smart buffer (New England Biolabs).

Enzymes (New England Biolabs).
Alu1
Mse1
Xcm1
Nco1
PCR water
cDNA
Autoclaved PCR tubes
Incubator at 37C
2% Agarose gel

Preparation of master mix (10 l)

cDNA DNA - 3 l
10 X NEB cut smart buffer
PCR water

- 1 l

- 5 l

Master mix was prepared 4 tubes and mixed well

9 l of master mix was aliquoted in each tube.
1 l of restriction enzyme was added in the respective tubes carefully and mixed well.
Tubes were kept at 37C for 16 h.
Samples were loaded along with DNA marker (NEB 100 bp ladder) on 2.5 % agarose gel and

electrophoresed at 120 V.
Gel was stained with ethidium bromide solution and gel image was documented.
18

2.10. Restriction digestion for cloning

Restriction digestion of purified PCR product and pET28a plasmid with Nde I and EcoR I.

cDNA (Os03g22810) - 20.0 l

Plasmid (pET28a)-

20.0 l

NEB cut smart buffer- 5.0 l

NEB cut smart buffer - 5.0 l

Nde I -

2.5 l

Nde I -

2.5 l

EcoR I -

2.5 l

EcoR I-

2.5 l

Water -

20.0 l

Water -

20.0 l

Total -

50.0 l

Total -

50.0 l

Samples were kept at 37C for 6 h.

Digested products were 1% agarose gel and stained with ethidium bromide solution.
Digested fragments of PCR and plasmid were cut from agarose gel using sterile surgical

blade.
DNA was purified from gel pieces using gen elute EtBr spin (sigma).

2.11. Ligation
Digested pET28a and Os03g22810 cDNA was ligated using T4 DNA ligase.
Vector (pET28a) -

14.0 l

Insert (cDNA) -

2.0 l

10 X buffer-

2.0 l

T4 ligase- `

2.0 l

Total-

20 l

All the components are mixed well and incubated at room temperature for 1 h.

2.12. Purification of ligation product

19

300 l of distilled water was added in the ligation mix followed by addition of 200 l of
phenol: chloroform: isoamyl alcohol (25:24:1), mixed and centrifuged at 12000 rpm for 10

min at 4C.
Aqueous phase was transferred in to a fresh tube.
1 l of LPA (Linear poly acryl amide), 10 l of sodium acetate and 100 % ethanol was added

in to aqueous phase.
Tube was kept in - 20C for overnight for precipitation.
Tube was centrifuged at 14000 rpm for 30 minutes and supernatant was discarded.
Pellet was washed with 70 % ethanol and centrifuged for 14000 rpm for 15 minutes.
Supernatant was discarded carefully and pellet was air dried and resuspended in 10 l of
distilled water.

2.13. Preparation of Bacterial stock:

A single colony of the E. coli was inoculated in LB- broth with appropriate antibiotics (if

required) and grown in shaker at 37C for 16 h.

200 l of 100 % glycerol was added in 1 ml of the bacterial grown culture and mixed

thoroughly. (Final glycerol concentration : 20 % glycerol)

Culture was kept in -70C for long term storage.

2.14. Preparation of electro competent cells

Bacterial strain: E. coli DH5
Protocol

A single colony of DH5 was inoculated in 5ml of L.B broth and kept in shaker at 37C for 1

hr.
1% incoulum of DH5 culture was transferred to 100 ml L.B broth and grown at 37C till

OD600 reached ~ 0.4-0.5

E. coli cells were harvested at 6000 rpm for 10 min at 4C.
Supernatant was discarded and remaining media was decanted by inverting the tube in tissue

paper.
50 ml of ice cold 10% glycerol was added to cell pellet and resuspended gently and

centrifuged at 7000 rpm for 10 min at 4C.

Supernatant was discarded and pellet was resuspended in 25 ml of ice cold 10% glycerol and

centrifuge at 7000 rpm for 10 min at 4C.

Again discard the supernatant and pellet was resuspended in 1 ml of ice cold 10% glycerol.
20

50 l of cells were adequate in sterilized 0.5 ml tubes and froze in liquid nitrogen.
Cells can be stored at -70C.

2.15. Transformation
Requirements:

Electroporator (Eppendorf)
LB broth and agar
Kanamycin (50 mg/ml)
Electroporation cuvettes

Protocol

Purified ligation, electroporation cuvettes and competent cells were put on ice.
3 l of purified ligation was added into 40 l DH5 E. coli cells in cuvettes and mixed

carefully.
Cells were transferred in electroporation cuvette and placed into electroporator, pulse of 2

kV cm-1 was given and 1 ml of LB broth was added immediately and mixed.
Culture was transferred to fresh tube and placed in shaker at 37C for 1 h.
Transformants were selected on LB-agar with kanamycin (50 g/ ml) by plating 100 l of

culture and kept in incubator at 37C for 16 h.

Colonies were patched on fresh LB-agar kanamycin plate and kept at 37C.

2.16. Screening of transformants

Positive clones were confirmed by colony PCR and restriction digestion.

Colony PCR:
10 X buffer

2.5 l

dNTPs

1.0 l

MgCl2

1.0 l

T7 (FP)

0.5 l

T7 (RP)

0.5 l

Taq DNA pol (5U/ l) 0.2 l

21

Water

19.5 l

Total

25 l

Master mix was prepared and aliquoted in different tubes.

Very small amount of colony was picked with fine needle and dipped into the solution

containing PCR tube and mixed for 2-3 times

Needle was sterilized by heating in flame till red hot, cooled and next colony was picked.
This cycle repeated for picking each colony.
All the samples were tap mixed and centrifuged.
Tubes were placed in PCR machine with following given programme.

Colony PCR programme:

Initial denaturation: 94C - 10 min
Denaturation:

94C - 45 sec

Annealing:

60C - 45 sec

Extension:

72C - 1min 20 sec

Final extension:

72C - 10 min

Stored at

35 cycles

4C

Sample preparation

4 l of sample was mixed with 1 l of 5 X DNA sample loading dye.

Samples were loaded in 1.5 % agarose gel and electrophoresed at 100 V.
Gel was stained with ethidium bromide and gel image was documented.

2.17. Plasmid isolation

Plasmid isolation was done by alkaline lysis method.
Requirements:
i) Alkaline lysis solution :
25 mM Tris buffer (pH 8.0)
50 mM glucose
10 M EDTA (pH 8.0)
ii) Alkaline lysis solution II:
0.2 N NaOH
1% SDS
22

iii) Alkaline lysis solution III:

5 M potassium acetate
Glacial acetic acid
Distilled water
iv) 1X Tris-EDTA (TE) buffer pH 8.0
Protocol

Single clone was inoculated in 10 ml of LB kan and kept at 37C shaker for 16 h.
The culture was centrifuged at 7,000 rpm for 5 min at 4C and discarded the flow through.
Pellet was resuspended in 200 l of ice-cold alkaline lysis solution I by vortexing.
400 l lysis solution II was added and gently mixed by inverting the tubes and kept for 5 min

at 4C.
300 l lysis solution III was added and mixed by inverting the tubes and kept for 5 min at

4C.
Centrifuged at 12,000 rpm for 5mins at 4C and collect the supernatant in fresh tube.
1 ml of phenol was added and centrifuged at 12,000 rpm for 5 min at 4C.
Aqueous phase was transferred in fresh tube and equal volume of chloroform: isoamyl
alcohol (24:1) was added and centrifuged at 12,000 rpm for 5mins at 4C.
Aqueous phase was isolated carefully into fresh tube and 1/10th volume of 3 M sodium
acetate and 2.5 volume of isopropanol was added, mixed and kept at 20C for 30 min.
Centrifuged at 12,000 rpm for 15 minutes and supernatant was discarded.
The pellet was washed with 70% ethanol and supernatant was discarded.
Pellet was air dried and dissolved in 1X TE buffer.
Samples were loaded in 1% agarose gel and electrophoresed at 100 V.
Gel was stained with ethidium bromide and gel image was documented.

2.18. Preparation of Chemical competent E. coli cells

Requirement:

i) E. coli strain: BL-21(DE3)

ii) 100 mM CaCl2

A single colony of BL-21 E. coli cells were inoculated into 5 ml LB broth and kept on shaker
at 37C for 16 h.

1 % inoculum was transferred to 100 ml of fresh LB- broth and kept at 37C.
23

As the culture OD600 reached ~ 0.4-0.5, culture was kept on ice for 15 min.

Cells were harvested at 6000 rpm for 10 min at 4C and supernatant was discarded.

Pellet was resuspended in of the culture volume ice cold 100 mM CaCl2 gently.

Kept for 10 min in ice.

Cells were again centrifuged at 6000 rpm for 10 min at 4C and supernatant was discarded.

Pellet was resuspended in of the culture volume ice cold 100 mM CaCl2 gently.

Resuspended cells were kept on ice for 1 h.

Centrifuged at 6000 rpm for 10 min at 4C and supernatant discarded.

Finally, cell pellet was resuspended in 1ml of 100 mM CaCl2.

100 l of cell suspension was aliquoted.

For storage, 10 % glycerol was mixed.

Cells were frozen in liquid N2 and kept in -70C.

Transformation:

BL-21(DE3) competent cells were ice-thawed.

1 l of recombinant plasmid DNA of Os03g22810 was added into the BL-21(DE3) cells and

mixed gently.
Cells were kept in ice for 10 min.
Cells were given heat-shock at 42C for 2 min and immediately kept in ice for 10 min.
1 ml of LB- broth was added and cells were grown in shaker at 37C for 1 h.
Cells were centrifuged at 6000 rpm for 5 min and media was discarded.
Cells were resuspended in 100 l of LB- broth and plated on LB-agar kanamycin plate.
Plate was kept at 37C for 16 h.

2.19. Protein overexpression

Requirements:

LB- broth
Kanamycin stock ( 50 mg/ml)
Inducer: IPTG (isopropyl -D-1-thiogalactopyranoside)

Protocol:
24

A single colony of BL-21(DE3) containing pET28a-Os03g22810 was inoculated in 5 ml of

LB- broth containing 50 g/ml kanamycin and grown overnight at 37C in shaker.
1 % inoculum was transferred into 5ml of fresh LB-Kan broth in two tubes and grown further

at 37C.
As the culture OD600 reached ~ 0.4-0.5, in one tube, 1mM IPTG was added to induce the
protein expression and no IPTG was added in the other tube taken as uninduced. (Culture

without IPTG was taken as control).

Both the tubes were kept at 37C in shaker for 16 h.

2.20. SDS- Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Preparation of reagents:
a) 30 % acryl amide solution: (100 ml)

29.2 gms of acryl amide and 0.8 % N-N bisacrylamide was dissolved in 50 ml of distilled

water.
Solution volume was made up to 100 ml with distilled water.

b) 4X separating buffer (1.5 M Tris, pH 8.8)

Tris - 18.15 g

Tris was dissolved in the 80 ml of distilled water and adjust the pH to 8.8
Solution volume was made up to 100 ml with distilled water.

c) 10% SDS
SDS 10g
Distilled water 100 ml
d) 10% APS
Ammonium persulphate 0.1 g
Distilled water 1.0 ml
e) Bromophenol blue (0.1%)
5 mg of bromophenol blue was dissolved in 5 ml of distilled water.
f) Catalyst
25

TEMED
g) 2 X sample buffer
Tris (0.5M, pH 6.8) - 2.5 ml
SDS (10%)

- 4.0 ml

Glycerol (100%)

- 2.0 ml

mercvaptoethanol 0.8 ml
bromophenol blue
Distilled water

- 300 l
- 10 ml

h) Tank buffer
Tris 6.05 g
Glycine 28.80 g
10% SDS 10 ml
Distilled water 1000 ml
i) Staining solution (200 ml)
Coomassie blue (R-250) - 300 mg
Methanol

- 80 ml

Glacial acetic acid

- 20 ml

Distilled water

- 100 ml

Dye was 1st dissolved in methanol followed by acetic acid and water was added and

mixed.
Solution was filtered through Whatman filter paper No.1 and stored in brown bottle.

j) Distaining solution (1000 ml)

Acetic acid

- 100 ml

Methanol

- 300 ml

Volume of the solution made up to 1000 ml with distilled water.

Casting the gel:

Glass plates were cleaned with soap solution and washed with water thoroughly.
Plates were further cleaned with 70 % ethanol solution using lint free tissue paper.
Glass plate set was arranged in the gel casting tray.
26

Separating gel solution was prepared and TEMED was added in the last.
As soon as TEMED was added solution was mixed and immediately poured into the gel
cassette without creating air bubble.( Small amount of solution was left in the tube, to check

the polymerization is complete)

Layer of isopropanol (~ 200 l) was added on top of the gel for efficient polymerization.
After polymerization was complete isopropanol was removed using tissue paper.
Gel comb was placed between the plates, and stacking gel (4 %) was poured immediately
after adding TEMED. .( Small amount of

solution was left in the tube, to check the

polymerization is complete)
Allowed the gel to polymerization.
Stacking gel (4%):

Separation gel (18 %):

1 M Tris pH 6.8 - 0.625 ml

.5 M Tris pH 8.8-

3.0 ml

30% acryl amide- 0.850 ml

30% acryl amide

7.2 ml

10% APS

0.050 ml

10% APS

0.12 ml

10 %SDS

0.050 ml

10 %SDS

0.12 ml

TEMED

0.005 ml

TEMED

0.005 ml

Water

1.56 ml

Water

3.4 ml

Total

5 ml

Total

12 ml

200 l of both the cultures i.e. uninduced and induced were taken and centrifuged at 9000

rpm for 2 min, medium was discarded.

Cell pellet was resuspended in 10 l of water and mixed with 2X SDS-PAGE sample loading

dye.
Samples were then boiled in boiling water bath for 5 min and centrifuged at 10,000 rpm for 5

min.
Samples were loaded on the 18 % SDS-PAGE and run at 100 V.
Gel was stained with Coomassie brilliant blue R-250 solution (staining solution) and after

that destained with destaining solution.

Gel image was documented in gel documentation system.

3. Result
3.1. Plant growth in control and stress:
27

Rice genotype NSICRc 106 seedlings were grown in Hoagland media and
salinity stress treatment was given by supplementing the Hoagland media with

150 mM NaCl.
Effect of salinity of growth of the rice seedlings were shown in Figure 1.

Day 0

Control

Day 1

Stress

Control

Day 3

Stress

Control

Stress

Figure 1 Effect of salinity on rice growth

Growth of the rice was measured as shown in table 3.

Day 6

Day 7

Day 8

Day 9

Control

18.8 0.3 cm 25.2 0.8 cm

28.2 0.7 cm

30.1 0.5 cm

Stress

18.5 0.5 cm 17.7 0.5 cm

19.3 0.4 cm

20.5 0.6 cm

Table 3 Plant growth in control and stress

Results: Salinity stress retarded the growth of the rice plant and wilting of the
leaves was observed.

28

3.2. RNA extraction

Total RNA was isolated by Trizol method.

RNA was DNase treated for any DNA contamination.
5 l of RNA was loaded in 1% agarose gel ( to check the integrity of the

isolated RNA)
Gel was stained in ethidium bromide solution.
Gel image was documented and shown below: ( C = control and S = stress
sample RNA)
Day

0
C

1
C S

2
C

3
C S

4
C S

Figure 2. RNA Extraction

Result: Integrity of the RNA was good for cDNA synthesis.

3.3. RNA quantification

Quantification of RNA was done by UV spectrophotometer.

5 l of RNA sample was mixed with 995 l of DEPC water and absorbance was
measured at 260 nm.

1 OD260 for single stranded RNA = 40 g/ ml

Table 4 RNA quantification

29

S. No.

Name of Sample

OD260/5 l of stock RNA

RNA concentration

Concentration

(A)

(g/ 5 l) = OD260 x 40

(g / l) = (B)/5

(B)

1
2
3
4
5
6
7
8
9

0 day control
1 day control
1 day stress
2 day control
2 day stress
3 day control
3 day stress
4 day control
4 day stress

0.183
0.148
0.147
0.170
0.100
0.161
0.135
0.205
0.126

7.32
5.92
5.88
6.8
4
6.44
5.4
8.2
5.04

1.464
1.184
1.176
1.36
0.8
1.288
1.08
1.64
1.008

3.4. Complementary DNA (cDNA) synthesis

Total rice RNA (10 g) was reverse transcribed using SuperScript II reverse
transcriptase and oligo dT (N23) primer. The stock cDNA was diluted before PCR.

3.5. PCR amplification Cu/Zn Os03g22810 cDNA:

Full length of Os03g22810 cDNA was PCR amplified from total rice cDNA using

gene-specific primers and proof-reading DNA polymerase (Pwo DNA polymerase)

PCR Programme used as mentioned in materials and methods.
4 l of PCR product and 2 l of sample loading dye was mixed and loaded in 2.5%

of agarose gel.
Electrophoresed and staining in ethidium bromide solution and documented using
gel documentation system.

Gel image is shown below

M (bp)

1200
1000
500
400
300
200
100

Os03g22810 (485 bp)

30

Figure 3 PCR amplification of Os03g22810 cDNA

Observation:
Gel image showed the PCR amplification of 485 bp cDNA.
Result: Full length (485 bp) Os03g22810 cDNA was successfully PCR amplified.

3.6. Restriction digestion confirmation of purified PCR product:

3 l of purified PCR product was digested with Alu I, Mse I, Xcm I and Nco I
separately.

Kept for 16 h at 37C.

Samples were loaded along with DNA marker (NEB 100 bp ladder) on 2.5 %
agarose gel and electrophoresed.

Gel was stained in ethidium bromide solution and gel image was documented.

M (bp)

AluI MseI XcmI NcoI

1200
1000
517
400
300
200
100

31

Figure 4 Restriction digestion analysis of Os03g22810

Result: Restriction digestion of the PCR product showed the expected size fragments
thereby confirming that the amplified PCR product is indeed Os03g22810 cDNA.

3.7. Cloning of Os03g22810 cDNA

a) Restriction digestion and ligation:

Restriction digestion of Os03g22810 and pET28a was performed with Nde I and

EcoR I at 37C for 6 h.

Digested products were gel purified.
Purified digested DNA of pET28a and Os03g22810 was ligated using T4 DNA

ligase.
Ligation product was purified.

32

Figure 5.
pET28a (+)
plasmid map

b) Transformation:

5 l of ligation mix was added to DH5 E. coli electrocompetent cells.

Transformation was done by electroporation.
Cells were grown in LB broth and 100 l of culture was plated on LB-kanamycin
plate.

Figure 6: DH5 E. coli pET28a Os03g22810 transformants

33

3.8. Screening of transformants

a) Colony PCR:

Colonies were used as template in colony PCR.

T7 forward and T7 reverse primers were used for confirmation of positive clones.
PCR was done as mentioned in materials and methods.
5 l PCR products were loaded on 1.5 % agarose gel along with x 174 DNA Hae

III digested as marker and electrophoresed.

Gel was stained in ethidium bromide.
Gel image is given below. M = DNA marker x 174 Hae III, C = colony number)

Colony numbers

M C1 C2

C3 C4 C5

C6 C7 C8

C9

Marker (bp)

1353
872
603

765 bp

310

Figure 7 Colony PCR of the transformants

Expected size of the PCR products amplified from positive clones: 765 bp

Positive clones are identified in colony PCR (C2, C4, C6, C7, C8, and C9).

C1, C3 and C5 are negative clones and not contain our gene of interest.

Result: Six positive clones C2, 4, 6, 7, 8 and 9 obtained thorough Colony PCR
confirmation.

3.9. Plasmid isolation

34

Plasmid was isolated by alkaline lysis method

Plasmids were isolated from positive clones (C2, C4, C6, C7, C8, and C9).
Plasmid samples were stored in - 20C.
2 l of sample was loaded in 1% agarose gel and stained in ethidium bromide

solution.
Gel image was shown below:
1

Figure 8. Plasmid DNA isolation

4.0. Transformation and Protein overexpression in E. coli:

Recombinant plasmids pET28a-Os03g22810 cDNA of clone 2, 4 and 6 were

transformed into chemical competent E. coli BL-21 (DE3) cells as mentioned in

materials and method.

Overnight cultures of BL-21(DE3) pET28a-Os03g22810 was induced with 1mM

IPTG for 8 h at 37C.

1 ml of the bacterial culture was mixed with 20 % glycerol and stored in -70C.
Cells were pelleted by centrifugation and lysed as described in material and method.
Supernatant (cell lysate) was loaded in 18 % SDS- PAGE, electrophoresed followed

by coomassie staining.
Gel image was documented.

C2

C4

C6

35
Os03g22810

UI

UI = Uninduced, C2, C4 and

C6

Clone

numbers,

Induced)

Figure 9: Overexpression of Rice Cu/Zn SOD Os03g22810

Result: Rice Cu/Zn Superoxide dismutase Os03g22810 protein was successfully overexpressed

in E. coli.

36

4. Discussion
Salinity tolerance, a complex phenomenon, involves contribution from several
molecular mechanisms that provide protection against different kinds of salt induced
cellular damage viz. ion toxicity, osmotic stress, oxidative stress etc. (Sreenivasulu et
al. 2007; Munns and Tester, 2008). Efficacy of such mechanisms (in minimizing salt
induced damage) vary among genotypes of a species and depends upon several factors
such as, presence/absence stress responsive genes/proteins and their basal level (Taji et
al. 2004) and how rapidly they are modulated under stress (Kawasaki et al. 2001).
The present study was focused on understanding the involvement of Cu/Zn SODs in
minimizing the salinity oxidative stress in rice seedlings. As mentioned in the
introduction plants being sessile are more prone to such stress induced damage and
harbor several enzymatic and non-enzymatic mechanisms to contain ROS induced
cellular damage (Karuppanapandian et al. 2011).
Cu/Zn SODs, the ubiquitous metalloenzymes, constitutes the first line of defense
against oxidative damage (Scandalios 1990; Gill and Tuteja 2010) which constitutes an
important component of most abiotic and biotic stress conditions and if not contained
results in extensive cellular damage (Mittler 2002; Langebartels et al. 2002). Cu/Zn
SODs, the ubiquitous metalloenzymes, constitutes the first line of defense against
oxidative damage (Scandalios 1990; Gill and Tuteja 2010) which constitutes an
important component of most abiotic and biotic stress conditions and if not contained
results in extensive cellular damage (Mittler 2002; Langebartels et al. 2002).
In rice, there are four Cu/Zn SODs which are localized in different cellular
compartments and are also regulated differentially in physiological conditions as well
as under stress responses. All are collectively involved in minimizing the stress induced
oxidative stress. In rice Cu/Zn SOD isozymes are major contributors to total SOD
activity in different physiological stages (Pan and Yao 1992) and cytosolic Cu/Zn SOD
the genes were found to be responsive to abiotic stress (Sakamato et al. 1992; Pan et al.
1995). The rice Cu/Zn SODs have also been extensively studied at transcript level,
however not all of them have been characterized at protein level. Pan et al. (1999)
37

cloned the rice cytosolic Cu/Zn SOD, and expressed in E. coli, for biochemically
characterization. All the Cu/Zn SODs have not been extensively studied.
In this project, full length cDNA of rice Cu/Zn SOD encoded by locus Os03g22810 of
rice genome was isolated, followed by cloning in a plasmid vector for heterologous
expression of the recombinant protein. These objectives were completed within the
timeframe of the project. Later the protein will be purified and its biochemical
characteristics will be studied.
The very first part of the experiment was to grow the rice seedlings, hydroponically, in
a standard Hoaglands liquid growth media rice genotype under control conditions till
early seedling stage (6-day old plants). The rice seedling were germinated and later
grown in a plant growth chamber under control light, temperature and humidity
conditions. One set of seedlings was shifted to growth media containing 150 mM NaCl
for salinity treatment. Both the sets were recorded for seedling growth everyday till 9 th
day. The salinity treatment substantially affected the growth of NSICRc106 genotype,
as salinity is known to affects every aspect of plant physiology, thus affecting the
growth. Within 1-day of salinity treatment the NSICRc106 seedlings show ~30%
reduction in growth compared to control seedlings.
To carry out any gene expression studies by northern blotting, semi-quantitative reverse
transcriptase PCR (RT-PCR), and quantitative PCR (qRT-PCR) etc., a good quality of
total RNA is a pre-requisite. Total RNA of rice seedlings at different time-points was
isolated using a combination of Qiagen homogenizer and Trizol method. Qiagen
homogenizer prevents the contamination of RNase in tissue samples, simplifies the
procedure and gives similar quality and quantity, whereas Trizol reagent offers a simple
procedure for rapid isolation of good quality RNA. This was evident in the RNA
preparations of nine rice samples. The samples show good quality and quantity for
required for further processing for cDNA synthesis.
For synthesis of complementary DNA, the total RNA was used. Total RNA also
contains messenger RNA (mRNA), which has polyA-tail at the 3-end in eukaryotes.
This gives advantage for their purification and selective cDNA synthesis. This property
was used for cDNA synthesis using oligo-dT primer (which binds to the polyA-tail of
38

mRNAs) and a reverse transcriptase enzyme, SuperScript II. This step covert mRNA
into complementary strand of DNA, referred to as cDNA. This step is also crucial and
care is should avoid contamination of RNase, or else there will no or very less cDNA
generated. Hence all reagents and plastic consumables used in this step were RNasefree. The product thus obtained was then converted into double stranded DNA molecule
by PCR using Pwo polymerase. This enzyme is a proof reading polymerase and has
less mutation frequency which is desirable when working with PCR amplification to
amplify genes for functional characterizations.
The approach was useful and amplified full length cDNA of the rice cytosolic Cu/Zn
SOD Os03g22810.
Any PCR product when amplified should also be sequenced, as the expected length
does not mean the perfect sequence. One of the preliminary way to quickly assess the
PCR product is by restriction analysis. The cDNA sequence available in the rice
genome database was analyzed in silico for restriction sites and some of them were
selected and later used actually to digest the PCR amplified cDNA of the rice Cu/Zn
SOD. The restriction digestion pattern indicated that the PCR product is indeed the
expected product. The full sequence of the product is in progress. The cDNA fragment
was then cloned in pET28a using a strategy based on NdeI and EcoRI enzymes for
digestion of plasmid and the insert. This strategy offers direction cloning and less
number of false positive clones. The ligation mix was purified and transformed into
DH5 E. coli cells by electroporation, a high efficiency method. Several positive clones
were identified, transferred to another plates and later confirmed colony-PCR method.
Colony PCR is a quick method to ascertain the presence of insert in the plasmid after a
cloning experiment. Here, the primers used were specific to the pET28a plasmid and
the results yielded expected sized PCR products in several clones. These positive clones
were then used for high quality plasmid isolation for sequencing and protein over
expression in another E. coli host, BL-21 cells.
As the E. coli DH5 cells are suitable for cloning of genes and not for heterologous
over expression of proteins, the purified plasmid was transformed in BL-21 cells. Bl-21
E. coli strain is suitable for overexpressing large amount of recombinant protein, which
39

can be purified and used for subsequent analysis. The cells were induced by IPTG to
overexpress protein at 37C. Preliminary analysis showed that the recombinant rice
cytosolic Cu/Zn SOD is over expressed in E. coli. The protein can be now used for
various studies to understand it biochemical and biophysical characteristics. However,
this is not sufficient as there is a common problem of improper folding and aggregation
during heterologous over-expression proteins in E. coli (Evans and Xu 2011). Hence,
the conditions will be further optimized to get more amount of recombinant protein. In
addition, the protein should be in soluble form and not mis-folded or aggregated.
This project could be successfully isolated and cloned the full length cDNA in an
expression vector. The protein was also successfully over expressed in E. coli cells.
Future studies will be focused on the purification and analysis of the protein, which will
reveal the importance of the Cu/Zn SOD in scavenging the superoxide radicals and
minimizing the cellular damage due to oxidative stress.

40

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