A study on relationship among five different varieties of

Nerium oleander L. through SDS PAGE

Keywords:
Nerium oleander, SDS-PAGE, Protein variability
ABSTRACT:




This study was done to analyze the relationship between five different
varieties of Nerium oleander belonging to the family Apocynaceae collected from
Tirunelveli District of Tamilnadu. The five varieties of the plant species were
characterized by analyzing protein variability in leaf tissues through SDS PAGE. The
electrophorogram showed a total of 45 bands of proteins with molecular weight
ranging from 4.29 to 160.94 KDa. The protein diversity analysis was done based on
the presence and absence of bands thereby interpreting their relatedness. The cluster
analysis clearly showed that there was high degree of diversity among these varieties
of plant species.

007-011 | JRPS | 2011 | Vol 1 | No 1
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www.plantsciences.info
Journal of Research in
Plant Sciences
An International Scientific
Research Journal
Authors:
John De Britto A, Steena
Roshan Sebastian, Mary
Sujin R and Jeba J.


Institution:
Plant Molecular Biology
Research Unit,
PG & Research Department
of Plant Biology and
Biotechnology,
St.Xavier's College,
(Autonomous),
Palayamkottai - 627 002,
Tamilnadu, India.


Corresponding author:
John De Britto A


Email:
bjohndesxc@gmail.com








Phone No:
0091- 462 4264374



Fax: 0091- 462-2561765




Web Address:
http://plantsciences.info/
documents/PS0005a.pdf.


Dates:
Received: 04 Nov 2011 /Accepted: 08 Nov 2011 /Published: 11 Nov 2011
Article Citation:
John De Britto A, Steena Roshan Sebastian, Mary Sujin R and Jeba J.
A study on relationship among five different varieties of Nerium oleander L. through
SDS PAGE
Journal of Research in Plant Sciences (2011) 1: 007-011
An International Scientific Research Journal
Original Research Paper
Journal of Research in Plant Sciences
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INTRODUCTION
All taxonomists agreed that the differences
between plants and the similarities that plants may
possess in common are measurable to large degree
by the morphological characters of those plants
(Lawrence, 1971). When the plants are highly
variable and contain large number of hybrids,
identification based on morphological characters is
quite difficult. Modern biological techniques are
now available that can resolve systematic
relationships, inter specific and intra specific
studies (Karihaloo et al., 2002).
Among several biochemical techniques,
SDS-PAGE (Sodium Dodecyl Sulphate) has been
used successfully to resolve taxonomic and
evolutionary problems of several plants (Khan,
1992; Rabbani et al., 2001). In this technique
protein is separated according to their molecular
weights. Resolution of this technique is very high
and therefore it could be used as a reliable tool for
taxonomic purposes (Bartke et al., 1966). Its
banding pattern is very stable which advocated for
identification purpose in medicinal plants. It has
been widely suggested that such banding patterns
could be used as important supplemental method
for medicinal plant identification (Tanksley and
Jones, 1981; Thanh and Hirata, 2002). Analyses of
SDS-PAGE are simple and inexpensive, which are
added advantages for its use in practical plant
breeding technique (Elham et al., 2010).
Nerium oleander L. belonging to the family
Apocyanaceae is widely distributed in the
Mediterranean and subtropical Asia. Although used
principally as an ornamental bush, its medicinal and
toxicological properties have also been recognized.
It is used in the treatment of a wide variety of
maladies and conditions, including abscesses,
corns, asthma, dysmenorrhea, eczema, epilepsy,
herpes, malaria, psoriasis, ringworm, scabies, sores,
warts, and certain tumors (Langford and Boor,
1996).
Different varieties of N. oleander exhibited
variability which is identified morphologically with
varying colour of the flowers. Hence an attempt
was made to study the variability through SDS
PAGE.

MATERIALS AND METHODS
The leaves of Nerium oleander L. with pink
(N
1
), red (N
2
), white (N
3
), dark pink (N
4
) and
rose (N
5
) colour flowers were collected from a
medicinal garden in Tirunelveli district of
Tamilnadu. Young leaves were taken in plastic bags
for transport from the field to laboratory. The
samples were maintained in deep freezer at -70 C.
The separation of protein was carried out in 50 C at
100V thereafter for 3-5 hours. SDS-PAGE
electrophoresis preparation was followed by
Laemmli (1970) and then the molecular weight of
unknown protein was determined. The NTSYS and
TL 100 softwares were used for the calculations and
analysis.

RESULTS AND DISCUSSION:
Proteins isolated from the leaves of five
different varieties of N. oleander were subjected to
SDS-PAGE analysis and observed a total of 45
bands (Plate 1). Highest number of bands (13) was
found in N
3
with white coloured flowers, while least
number of bands (7) were found in N
2
and N
5
with
red and rose coloured flowers respectively.
Using Total Lab 100 protein analyzer
software the molecular weight and the Rf value of
each band were calculated. The molecular weight of
proteins ranged from 4.29 KDa to 160.94 KDa
isolated from the leaf tissues of five varieties of
Nerium oleander (Table 1). The least molecular
weight protein (4.29 KDa) was present in N
3
with
highest Rf value 0.97 and highest molecular weight
protein (160.94 KDa) were also found in N
3
with
lowest Rf value 0.13 (Table 1).
NTSYS pc software was used to analyze the
phylogenetic relationship based on protein pattern.
The similarity coefficient was calculated on the
basis of presence and absence of bands which
ranged from 0.530.88 (Table 2), and a UPGMA
dendrogram was constructed using the similarity
coefficient (Fig. 1). The clusters obtained from the
Britto et al.,2011
008 Journal of Research in Plant Sciences (2011) 1: 007-011
Fig. 1: Cladistical analysis of Protein samples of five
different varieties of Nerium oleander
dendrogram showed that the studied five varieties
of N. oleander were grouped into two main clades,
C
1
and C
2
. The C
1
is again subdivided into two
subclades C
1
S
1
and C
1
S
2
, of which the C
1
S
1
is
further subdivided into two divisions, C
1
S
1
a and
C
1
S
1
b. The C
1
S
1
a holds N
1
and N
2
with a similarity
of 64%. C
1
S
1
b having N
5
variety formed a separate
clade with 50% similarity. C
1
S
2
clade holds N
3
and
N
4
varieties with a similarity of 68%. All these five
varieties diverge from a common origin with about
38% similarity.

Britto et al.,2011
Journal of Research in Plant Sciences (2011) 1: 007-011 009
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Similar study was done by Syed et al.,
(2008) in Elaeagnus umbellata (Thunb.) that
belongs to the family Elaeagnaceae. The seeds of
eight ecotypes from Azad, Jammu and Kashmir,
Pakistan, were analyzed to study their relationship
and evolution based on SDS-PAGE of total seed
proteins. The results indicated that each population
can be distinguished by their own specific protein
bands with reference to a molecular weight marker
included in the gel. The dendrogram based on
computer package analysis indicated that
populations having the same base of origin fall
under two simultaneous groups, i.e. P
6
and P
8
in one
group and P
1
, P
2
, P
3
, P
4
, P
5
, and P
7
in the second
group. SDS-PAGE thus provided valuable
information for the identification of populations and
could be utilized for population and varietals
discrimination as well as seed quality test in true to
type seed producing plants; Phylogenetic
relationship and similarity indices of some Acacia
species using SDS-PAGE gel electrophoresis was
done by Somia and El-akkad (2004); Phylogenetic
diversity of some tomato varieties was done by
Elham et al. (2010).
This work was framed to study the
molecular variability on the basis of protein profile
and to establish their phylogenetic relationship with
one another.

CONCLUSION
In the present study an attempt has been
made to analyze the protein variability among five
varieties of Nerium oleander with variedly coloured
flowers and it is clear that all the five varieties had
varied differences in their protein banding pattern.
Based on their protein banding pattern, the
phylogenetic tree constructed shows that the
varieties are more or less closely related to each
other. Further analysis using molecular markers will
help to understand the genetic variability of these
varieties.

REFERENCES
Bartke EM, Watt DD and Tu T. 1966.
Electrophoretic pattern of venoms from species of
crotalidae and elapidae snakes. Toxicol., 4:73-76.

Elham AA, Abd El-Hady, Atef Haiba AA,
Nagwa R, Abd El-Hamid and Aida Rizkalla A.
2010. Phylogenetic Diversity and Relationships of
Some Tomato Varieties by Electrophoretic Protein
and RAPD analysis. J. Amer. Sci., 6(11):434-441.

Karihaloo JL, Kaur M and Singh S. 2002. Seed
protein diversity in Solanum melongena L., and its
wild and weedy relatives. Genetic Res. and Crop
Eval., 49(6):533-539.

Khan MA. 1992. Seed protein electrophoretic
pattern in Brachypodium P. Beavu. Species. Ann.
Bot., 70:61-68.

Laemmli UK. 1970. Cleavage of structural proteins
during the assembly of the head of bacteriophage
T4. Nature 227:680-685.

Langford SD and Boor PJ. 1996. Oleander
toxicity: an examination of human and animal toxic
exposures. Toxicol., 109:1-13.

Lawrence GHM. 1971. Taxonomy of the vascular
plants. The Macmillan Company, New York. 693-
695.

Rabbani MA, Quershi AS, Azfal M, Anwar R
and Komatsu. 2001. Characterization of mustard
(Brassica juncea (L.) Czern. & Coss.) germplasm
Britto et al.,2011
010 Journal of Research in Plant Sciences (2011) 1: 007-011
Varieties N
1
N
2
N
3
N
4
N
5

N
1
1.00
N
2
0.61 1.00
N
3
0.44 0.53 1.00
N
4
0.30 0.60 0.66 1.00
N
5
0.40 0.57 0.16 0.28 1.00
Table 2: Similarity Index of five different varieties of
Nerium oleander based on SDS-PAGE
by SDS-PAGE of total seed proteins. Pak. J. Bot.,
33(2):173-179.

Somia S and El-akkad. 2004. Phylogenetic
Relationship and Similarity Indices of Some Acacia
Species Using Seed Protein Analysis. Int. J. Agri.
Biol., 3:435-439.

Syed DA, Syed M, Sabir D, Halimi MS and
Yousaf S. 2008. Evolutionary Relationship and
Divergence Based on SDS-PAGE of Elaeagnus
umbellata Thunb. Populations, a Multipurpose
Plant from the Himalayas. Turk. J. Biol., 32:31-35.

Tanksley SD and Jones RA. 1981. Application of
alcohol dehydrogenase allozymes in testing the
genetic purity of F1 hybrids of tomato. Hort.
Sci.16:179-181.

Thanh VOC and Hirata Y. 2002. Seed storage
protein diversity of three rice species in the Mekong
Delta. Biosphere Conserv., 4:59-67.
Britto et al.,2011
Journal of Research in Plant Sciences (2011) 1: 007-011 011
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