To investigate the phylogenetic relationship of nine Ipomoea species, seed proteins were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Based on the analysis, total 50 bands were identified. The number of bands varies from 8 bands in Ipomoea obscura to 4 in Ipomoea mauritiana. Phylogenetic tree was constructed based on the presence or absence of protein bands using Freetree and Treeview software programme

Pragati, V.G.Parameshwar.

Sreenath, K.P.

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 International Research Journal of Biological Sciences ___________________________________ ISSN 2278-3202 Vol. 2(10), 64-67, October (2013) Int. Res. J. Biological Sci.   International Science Congress Association        64 Phylogenetic Relationship of Some Ipomoea Seed Proteins by SDS-PAGE  Pragati V.G. Parameshwar and Sreenath K.P. Department of Botany Bangalore University, Bangalore, Karnataka 560056, INDIA  Available online at: www.isca.in, www.isca.me Received 14th September 2013, revised 3rd October 2013, accepted 9th October 2013    Abstract  To investigate the phylogenetic relationship of nine Ipomoea species, seed proteins were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE).  Based on the analysis, total 50 bands were identified.  The number of bands varies from 8 bands in Ipomoea obscura to 4 in Ipomoea mauritiana. Phylogenetic tree was constructed based on the presence or absence of protein bands using Freetree and Treeview software programme.  Keywords: Ipomoea, protein, phylogenetic tree, bands, SDS-PAGE  Introduction The convolvulaceae (Morning Glory Family) is a beautiful family which is widely cultivated as ornamentals. About 55 genera and 1930 species of the convolvulaceae are widely distributed throughout temperate and tropical regions and abundant in tropical America and tropical Asia1. One of the major genus is Ipomoea, represented by 600 species2.  There are number of sections in the genus Ipomoea3. The morphological traits and biochemical analysis they go hand in hand4. Phylogenetic analysis of 40 species representing the three subgenera and nine sections within the Ipomoea using sequence data from the ITS region and waxy sequences revealed a close relationship between species of section Pharbitis subgenus Ipomoea and species of subgenus Quamoclit5 The cladistic analysis of the tribe Ipomoeae based on 45 morphological and palynological characters, and suggested that the Ipomoeae is a monophyletic tribe6.  The phylogenetic relation of the genus Ipomoea with other genera from the tribe Ipomoeae based on morphology and phylogeny and found that Ipomoea is paraphyletic7.  The objective of our study is to clarify the relationship among the nine Ipomoea species using the molecular technique of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of seed proteins.  Material and Methods  Seeds of Ipomoea species were collected from different parts of Karnataka.  These Ipomoea species were used to study uni-dimensional SDS-PAGE (12.5% resolving gel and 4% stacking gel) in a vertical gel system8 (Bio-Rad).  Samples were prepared as follows. 0.2gm of seeds grounded in 2ml of 50mM phosphate buffer (pH 7.4) in cold conditions to get 10% of homogenate. Then it is centrifuged in micro-centrifuge machine for 10 minutes at 10,000 rpm.  The supernatant was separated and used as protein sample. The protein concentration in the supernatant was then determined for gel electrophoresis by the method of Bradford9, with bovine serum albumin (BSA) as the standard and using spectrophotometer at 595 nm. The protein sample along with the gel loading buffer containing bromophenol blue were denatured in boiling water (1 minute), cooled and 100 µg  of each extract loaded in lanes with micropipette. A protein molecular weight marker (Aristogene, Bangalore, Cat No. BCL-039) was also incorporated into the gel (as marker lane) as reference to detect molecular weights of the bands.  The gel was ran initially at 50mA for half an hour later on at 100mA for 2 hours.  Following electrophoresis, the gel was stained with a solution containing Coomassie brilliant blue (CBB-R-250), destained with double distilled water. All chemicals were purchased from Sigma and stock solutions were prepared before making a working solution. The gel was scanned using Alpha Innotech 1.2 version.  The presence or absence of each band was treated as binary character in a data matrix i.e coded 1 and 0 respectively.  Data were analyzed using Freetree software programme10 with Jaccard method11 and UPGMA (Unweight Pair-Groups Method using Arithmetic Average) by tree construction method using Treeview software12.  Results and Discussion The seed protein banding profile among nine species of Ipomoea is compared in figure 1. The zymogram of the same is presented in figure 2. Electrophoretic analysis of proteins exposed a total of 50 protein bands in the seeds of the 9 species of Ipomoea. The analysis of the results reveals that some bands are characteristic and constant markers for species.  Other bands are shared by more than one species.  The number of bands and intensity of bands varies from one species to another. The common band which is shared by all the plant species is at 55.5KDa (Rf value 0.655).  The Rf values, molecular weight, intensity and position of protein bands of Ipomoea species is given in table 1. The data International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202   Vol. 2(10), 64-67, October (2013)  Int. Res. J. Biological Sci.      International Science Congress Association  65 matrix of Ipomoea species showing the seed protein characters (0= band absent, 1= band present) is given in table 2. The phylogenetic tree obtained through Freetree and Treeview software figure 3, which indicates a very clear picture of the species inter-relationship.  The phylogram shows that the two groups of plant species seperated very early from each other and thus originated in seperate ways.  Eight plant species are in one group, only one species in another group. Further these 8 plant species forms two separate groups of one plant species  and seven plant species.  On the other hand, the 8 plant species, I. mauritiana, I. triloba, I. carnea, I. cairica, I. campanulata, I. obscura, I. hederifolia, I. muricata, originated from same ancestor shared by I. alba but seperated further into 2 groups represented by I. mauritiana in one group and I. triloba, I. carnea, I. cairica, I. campanulata, I. obscura, I. hederifolia, I. muricata,  in the other group during the process of evolution.   According to Biju sectional classification of the genus Ipomoea are Mina (I. hederifolia), Calonyction (I. muricata and I. alba), Erpipomoea (I. cairica and I. obscura), Eriospermum (I. carnea, I. mauritiana and I. campanulata) and Batatas (I. triloba) based on the morphological characters.The phylogenetic tree figure 3 constructed for 9 species of Ipomoea exhibit unrelatedness between the sections.  For example I. cairica and I. obscura are placed distantly, similarly I. alba and I. muricata exhibit far and wide relationship.  The I. triloba sandwiched between Eriospermum.  The section Mina and Calonyction go hand in hand, but I. alba is distinct. There is no correlation between SDS-PAGE profile and Biju’s morphological characters. The only possibility is to approach the species holistically.   Figure-1 Electrophoretic patterns of the seeds of Ipomoea Species:  M- Marker  (Wide range molecular weight standard from top to bottom 200, 97, 66, 45, 29 KDa.).  1- I. alba, 2- I. cairica, 3- I. campanulata, 4- I. carnea, 5- I. hederifolia, 6- I. mauritiana, 7- I. muricata, 8- I. obscura, 9- I. triloba.  Table-1 Rf values, molecular weight, intensity and position of protein bands of   Ipomoea species using  SDS-PAGE Band Rf value Mol.wt. in KDa. 1 2 3 4 5 6 7 8 9 1 0.310 155.8 + + ++ ++ - - - - - 2 0.345 119 - - - - - - - +++ - 3 0.500 72.6 - ++ +++ ++++ +++ + + ++++ + 4 0.569 66 +++ - - - ++++ - + ++++ - 5 0.655 55.5 +++ ++ ++++ ++++ ++++ ++ ++ ++++ +++ 6 0.724 45 +++ +++ ++++ ++++ ++++ +++ - +++ +++ 7 0.776 38.3 - +++ ++++ ++++ ++++ - ++ ++ +++ 8 0.862 29 + - - ++ - ++ - ++ - 9 0.914 14.5 + - + - - - + ++ ++ 1- I. alba, 2- I. cairica, 3- I. campanulata, 4- I. carnea, 5- I. hederifolia, 6- I. mauritiana, 7- I. muricata, 8- I. obscura, 9- I. triloba. (+: Low intensity, ++: Medium intensity, +++: High intensity, ++++: Very high intensity) International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202   Vol. 2(10), 64-67, October (2013)  Int. Res. J. Biological Sci.      International Science Congress Association  66  Figure-2 Zymogram of total soluble seed protein of Ipomoea species through SDS-PAGE    Figure-3  Phylogenetic tree obtained through banding comparisons among nine species of Ipomoea   International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202   Vol. 2(10), 64-67, October (2013)  Int. Res. J. Biological Sci.      International Science Congress Association  67 Table-2 Data matrix of Ipomoea species showing the characters (seed protein pattern) which is represented by fifty band, 0 = band absent 1 = band present                         Species Characters states     Ipomoea alba     Ipomoea cairica     Ipomoea campanulata     Ipomoea carnea     Ipomoea hederifolia     Ipomoea mauritiana     Ipomoea muricata     Ipomoea obscura     Ipomoea triloba 100111011 101011100 101011101 101011110  001111100  001011010  001110101  011111111  001011101  Conclusion Electrophoresis of seed proteins showed total of  8 bands in Ipomoea obscura, 6 bands in Ipomoea alba, Ipomoea campanulata, Ipomoea carnea, 5 bands in Ipomoea cairica, Ipomoea hederifolia, Ipomoea muricata, Ipomoea triloba, 4 bands in Ipomoea mauritiana.  The intensity of the band also varied among all the plant species.  Hence the present study obviously indicated the use of SDS-PAGE profile to draw inter-relatedness between the species of Ipomoea.  References 1. Saensouk S., The Family Convolvulaceae in Muang District, Nongkhai province Thailand, KKU Res. J., 12(3), 237-243 (2007) 2. Judd S.W., Campbell S.C., Kellogg A.E., Stevens F.P. and Donoghue J.M., Plant systematics: A Phylogenetic Approach 2nd ed. Massachusettes, USA:  Sinauer Associates,Inc. (2002)  3. Biju S.D., Taxonomic and morphologic studies on the family Convolvulaceae of southern peninsular India., PhD. thesis submitted to Calicut University, Kerala, (1997) 4. Das S. and Mukherjee K.K., Morphological and biochemical investigations on Ipomoea seedlings and their species interrelationships, Ann. Bot., 79, 565-571 (1997) 5. Miller R.E., Rausher M.D., Manos P.S., Phylogenetic systematics of Ipomoea (Convolvulaceae) based on ITS and waxy sequences, Syst Bot., 24, 209-227 (1999) 6. Wilkin P., A morphological cladistic analysis of the Ipomoeae (Convolvulaceae), Kew Bull, 54, 853-876 (1999) 7. Manos P.S., Miller R.E. and Wilkin P., Phylogenetic analysis of Ipomoea, Argyreia, Stictocardia and Turbina suggests a generalized model of morphological evolution in morning glories, Syst Bot., 26, 585-602 (2001) 8. Laemmli U.K., Cleavage of structural proteins during assembly of the head of bacteriophage T4, Nature, 227, 680-685 (1970) 9. Bradford M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding, Anal. Biochem., 72, 248-254 (1976) 10. Pavlicek A., Hrda S. and Flegr J., Free tree free ware program for construction of phylogenetic trees on the basis of distance data and bootstrap/Jack Knife analysis of the tree robustness, Application in the RAPD analysis of genus Frenkelia, Folia Biologiica., 45, 97-99 (1999) 11. Jaccard P., Nouvelles rescherches sur la distribution florale, Bull. Soc. Vaud. Sci. Nat., 44, 223-270 (1908)    12. Roderic D.C., Treeview 32, Versionl. Retrieved from: http:// taxonomy, zoology, gla. ac. uk. /rod/rod. Html., (2001)  

Phylogenetic Relationship of some Ipomoea Seed Proteins by Sds-Page

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Phylogenetic Relationship of some Ipomoea Seed Proteins by Sds-Page

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