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 Jou

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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 50º 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 N3 with white coloured flowers, while least

number of bands (7) were found in N2 and N5 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 N3 with highest Rf value 0.97 and highest molecular weight

protein (160.94 KDa) were also found in N3 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 0.53–0.88 (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,

C1 and C2. The C1 is again subdivided into two

subclades C1S1 and C1S2, of which the C1S1 is

further subdivided into two divisions, C1S1a and C1S1b. The C1S1a holds N1 and N2 with a similarity

of 64%. C1S1b having N5 variety formed a separate clade with 50% similarity. C1S2 clade holds N3 and

N4 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

Ref

B

an

d

Pin

k

Red

W

hit

e D

ark

pin

k

Rose

Nu

mb

er

Ba

nd

No

MW

(kd

) R

f B

an

d N

o

MW

(kd

) R

f B

an

d

No

MW

(kd

) R

f B

an

d

No

MW

(kd

) R

f B

an

d

No

MW

(kd

) R

f

1

1

15

6.2

3

0.1

3

- -

- 1

16

0.9

4

0.1

3

1

15

3.1

9

0.1

4

1

15

9.1

8

0.1

3

2

- -

- 1

14

7.5

1

0.1

5

2

14

7.9

9

0.1

5

- -

- 2

13

9.3

0

0.1

6

3

2

12

5.1

8

0.1

9

2

12

0.5

1

0.1

9

3

12

3.0

4

0.1

9

2

12

3.2

7

0.1

9

- -

-

4

3

11

3.0

2

0.2

1

- -

- 4

11

3.1

4

0.2

1

3

10

7.8

4

0.2

2

3

11

5.9

5

0.2

0

5

4

10

2.0

3

0.2

3

3

10

3.5

6

0.2

3

5

97

.28

0.2

4

4

91

.23

0.2

6

4

99

.79

0.2

4

6

5

75

.08

0.3

0

4

73

.94

0.3

1

6

82

.25

0.2

8

- -

- 5

75

.16

0.3

0

7

6

56

.19

0.3

7

5

60

.41

0.3

5

7

60

.81

0.3

5

5

53

.43

0.3

8

6

51

.22

0.3

9

8

- -

- 6

46

.14

0.4

2

8

44

.20

0.4

3

6

39

.55

0.4

5

- -

-

9

7

34

.28

0.4

9

7

32

.39

0.5

0

9

33

.23

0.4

9

- -

- 7

32

.65

0.5

0

10

8

28

.42

0.5

3

- -

- 10

22

.97

0.5

8

7

24

.35

0.5

7

- -

-

11

- -

- -

- -

- -

- -

- -

- -

-

12

9

11

.91

0.7

3

- -

- -

- -

- -

- -

- -

13

10

1

0.3

9

0.7

6

- -

- -

- -

- -

- -

- -

14

- -

- -

- -

11

8.5

3

0.8

1

8

8.3

5

0.8

1

- -

-

15

- -

- -

- -

12

5.6

1

0.9

1

- -

- -

- -

16

- -

- -

- -

13

4.2

9

0.9

7

- -

- -

- -

Tab

le 1

: M

ole

cu

lar w

eig

hts

an

d R

f va

lues

of

dif

feren

t va

rieti

es

of

Neri

um

ole

an

der

L.

N1-P

ink

; N

2-R

ed

; N

3-

Wh

ite;

N4-D

ark

pin

k;

N5-

Rose

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. P6 and P8 in one

group and P1, P2, P3, P4, P5, and P7 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

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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

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Khan MA. 1992. Seed protein electrophoretic

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Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage

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Langford SD and Boor PJ. 1996. Oleander toxicity: an examination of human and animal toxic

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Lawrence GHM. 1971. Taxonomy of the vascular

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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 N1 N2 N3 N4 N5

N1 1.00

N2 0.61 1.00

N3 0.44 0.53 1.00

N4 0.30 0.60 0.66 1.00

N5 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

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genetic purity of F1 hybrids of tomato. Hort.

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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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