Subscribe Now Subscribe Today
Abstract
Fulltext PDF
References
Research Article
 

Identification of Stevia rebaudiana Bertoni Proteins by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis



Ma Lei and Shi Yan
 
ABSTRACT

Four diverse genotypes of Stevia rebaudiana Bertoni were included for Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis. Total proteins were analyzed through slab type SDS-PAGE. Based on SDS-PAGE, specific bands were suggested to be used for identifying Stevia rebaudiana Berton. This method has the advantages of simplicity, high sensitivity and good accuracy and the SDS-PAGE proved to be a powerful tool for differentiating Stevia rebaudiana Bertoni varieties.

Services
Related Articles in ASCI
Similar Articles in this Journal
Search in Google Scholar
View Citation
Report Citation

 
  How to cite this article:

Ma Lei and Shi Yan, 2009. Identification of Stevia rebaudiana Bertoni Proteins by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis. Asian Journal of Crop Science, 1: 63-65.

DOI: 10.3923/ajcs.2009.63.65

URL: https://scialert.net/abstract/?doi=ajcs.2009.63.65

INTRODUCTION

Stevia (Stevia rebaudiana Bertoni, Compositae), a perennial herb native to Paraguay, is known to accumulate sweet diterpene glucosides such as stevioside and rebaudioside A mainly in the leaves. Stevioside, which is approximately 500 times as sweet as sucrose, is regarded as a valuable natural sweetening agent because of its relatively good taste and chemical stability (Yukiyoshi et al., 1984). Alternatives to sucrose serve a number of purposes. They are used to expand food and beverage choices for those who must or want to control caloric, carbohydrate, or sugar intake; assist weight control or reduction; aid in the management of diabetes; assist the control of dental caries; enhance the usability of pharmaceuticals and cosmetics; provide sweetness when sugar is not available; and assist the cost-effective use of limited resources (Cardello et al., 1999).

Two big problems can hardly be solved in Stevia rebaudiana Bertoni breeding and farming procedure, one is too long breeding period due to traditional breeding methods and the other one is stavioside content reduction due to impurity varieties (Tianhong and Yang, 1999; Motomu et al., 1994). Variety identification technique help to solve the above problems (Abdul et al., 2002).

Among biochemical techniques, Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) is most widely used due to its validity and simplicity for describing genetic structure of crop germplasm (Ahmad and Slinkard, 1992). Under the same growth conditions, leaf proteins can be used as genetic markers in four major areas: (1) analysis of genetic diversity within and between species, (2) plant domestication in relation to genetic resources conservation and breeding, (3) genome relationship and (4) as a tool in crop improvement. The SDS-PAGE is considered to be a practical and reliable method for species identification (Gepts, 1989).

One of the main aims of Stevia rebaudiana Bertoni cultivar identification is to use its specific genetic material for breeding programmes. The genetic distance between varieties can be assessed through morphological characterisation and genetic markers (Ahmad et al., 1996). The characterized material then helps the plant breeders to select the genotypes to be utilized in hybridization programme. The SDS-PAGE was implied to identify species of Stevia rebaudiana Bertoni which may be confused in the germplasm samples. In particular, the varieties which are confused and sometimes difficult to identify on the basis of morphological according to diagnostic techniques. Therefore, this situation suggested the need to have biochemical characterisation for identification of Stevia rebaudiana Bertoni species.

MATERIALS AND METHODS

The experiment was planted with two replications during Summer seasons of 2007 at Dryland Technology Key Laboratory of Shandong Province in Qingdao Agricultural University, Qingdao, China (36°N and 120° E).

Total protein were extracted from leaf of different diversities of Stevia rebaudiana Bertoni (No. 2 Shoutian from Japan, with high sugar, small leaf, low yield; No. 3 Xinguang bred by Jiangsu Yancheng Stevia Co. Ltd, with medium sugar, big leaf, high yield; No. 1 Qingtian bred by Qingdao Agriculture College, with high yield and disease-resistant; No. 2 Qingtian with significant difference from No. 1 Qingtian treated with Co-60 radiation, above species were planted in the same condition) with 100 mM Tris-Cl buffer (pH 8.0) containing 5 mM EDTA, 2% β-mercaptoethanol and 1 mM Phenyl Methyl Sulphonyl Fluoride (PMSF). The crude homogenate was kept for 45 min at 4°C and centrifuged at 11,000 rpm for 15 min. The extracted proteins were recovered as clear supernatant. Sample buffer (30 μL) was added to 30 μL of clear supernatant and mixed thoroughly in Eppendorf tube. The sample buffer (pH 6.8) contained the following final concentrations: 125 mM Tris, 20% glycerol, 10% β-mercaptoethanol, 0.01% Bromphenol Blue (BPB), 4% SDS. Bromophenol Blue (BPB) was added to the sample buffer as tracking dye to watch the movement of protein in the gel, the sample was boiled for 3 min before loading gel.

Total protein was analyzed through slab type SDS-PAGE using 5% upper gel and 15% lower gel. The SDS-PAGE of total protein was carried out in the discontinuous buffer system according to the method of Laemmili (1970) with a few modifications. The pH of lower gel was changed from 8.8 to 9.0 and the pH of upper gel was changed from 6.8 to 7.0 mL. In order to check the reproducibility of the method, two separate gels were run under similar electrophoretic conditions. Electrophoresis was carried out at a constant voltage of 100 V for 9 h. The gels were stained for 3 h in 0.1% (w/v) coomassie brilliant blue R-250 followed by destaining in ethanol : water : glacial acetic acid (1 :17 :2). Protein bands were visualized in a Tran illuminator under white light. Depending upon the presence or absence of polypeptide bands, differences between species was diagnosed for all possible pairs of protein types.

RESULTS AND DISCUSSION

The Stevia rebaudiana protein profiles were composed of approximately 25 bands. Most of the variation was observed in areas with mol in Fig. 1. The number of bands and position and definition of protein were different. Distinct quantitative variations were observed in the SDS-PAGE profile of total proteins of Stevia rebaudiana. The SDS-PAGE profile of total proteins of Stevia rebaudiana showed significant difference should be use for identification of variety.

This method has the advantages of simplicity, high sensitivity and good accuracy and this experiment proved that SDS-PAGE was a practical and reliable method for Stevia rebaudiana identification. It takes only 20 h from protein extraction to getting inspection reports and the SDS-PAGE is a fast and effective tool for differentiating Stevia rebaudiana Bertoni varieties based on the differences of protein components. Whereas a low level of inter-specific genetic diversity was observed for both the species and no clear differentiation was observed either for agronomic characteristics or their origin as various clusters consisted of mixed genotypes from different origins.

Fig. 1: (a) Variation in proteins of Stevia rebaudiana. The arrows indicate variation in different regions. 1: No. 2 Qingtian, 2: No. 1 Qingtian, 3: No. 2 Shoutian, 4: No. 3 Xingguang and (b) Fig. 1a was analyzed with the software of Quantity One and every peak responding a band and every peak corresponds with one band
REFERENCES
Ahmad, F. and A.E. Slinkard, 1992. Genetic relationships in the genus Cicer L. as revealed by polyacrylamide gel electrophoresis of seed storage proteins. Theoret. Applied Genet., 84: 688-692.
CrossRef  |  Direct Link  |  

Ahmad, M., D.L. McNeil and A.G. Fautrier, 1996. Genetic relationships in Lens species and parentage determination of their interspecific hybrids using RAPD markers. Theor. Applied Genet., 92: 1091-1098.
CrossRef  |  Direct Link  |  

Akita, M., T. Shigeoka, Y. Koizumi and M. Kawamura, 1994. Mass propagation of shoots of Stevia rebaudiana using a large scale bioreactor. Plant Cell Rep., 13: 180-183.
CrossRef  |  Direct Link  |  

Cardello, H.M.A.B., M.A.P.A. Da Silva and M.H. Damasio, 1999. Measurement of the relative sweetness of stevia extract, aspartame and cyclamate/saccharin blend as compared to sucrose at different concentrations. Plant Foods Hum. Nutr. (Formerly Qualitas Plantarum), 54: 119-130.
CrossRef  |  Direct Link  |  

Gepts, P., 1989. Genetic Diversity of Seed Storage Proteins in Plants. In: Plant Population Genetics, Breeding and Genetic Resources, Brown, A.H.D., M.T. Clegg, A.L. Kahler and B.S. Weir (Eds.). Sinauer Associates Inc., Sunderland, Massachusetts, pp: 64-82.

Ghafoor, A., Z. Ahmad, A.S. Qureshi and M. Bashir, 2002. Genetic relationship in Vigna mungo (L.) Hepper and V. radiata (L.) R. Wilczek based on morphological traits and SDS-PAGE. Euphytica, 123: 367-378.
CrossRef  |  Direct Link  |  

Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680-685.
Direct Link  |  

Tamura, Y., S. Nakamura, H. Fukui and M. Tabata, 1984. Clonal propagation of Stevia rebaudiana bertoni by stem-tip culture. Plant Cell Reports, 10: 183-185.
CrossRef  |  Direct Link  |  

Tianhong, C. and Z. Yang, 1999. Enrichment and separation of rebaudioside a from stevia glycosides by a novel adsorbent with pyridyl group. Sci. China ser. B, Chemis., 42: 277-282.
CrossRef  |  Direct Link  |  

©  2019 Science Alert. All Rights Reserved
Fulltext PDF References Abstract