Research Article | Volume: 5, Issue: 1, Jan-Feb, 2017

Expression analysis of recombinant Vigna radiata plant defensin 1 protein in transgenic tobacco plants

Hoang Thi Thao Nguyen Thi Ngoc Lan Ho Manh Tuong Nguyen Vu Thanh Thanh Le Van Son Chu Hoang Mau   

Open Access   

Published:  Jan 20, 2017

DOI: 10.7324/JABB.2017.50112
Abstract

Bruchid resistance is regulated by defensin gene. Vigna radiata plant defensin 1 (VrPDF1) inhibits alpha-amylase activities in insect gut; therefore, insect will die of undigestion starch. VrPDF1 content in mungbean seeds is very low. Hence an increase in content of VrPDF1 in mungbean seeds leads to enhance alpha -amylase inhibition in larvae and bruchids, which is necessary for the study to improve bruchid resistance in mungbeans. This article presents the results of VrPDF1 gene expression in T1 generation transgenic tobacco seeds. It was confirmed that VrPDF1 gene was attached to genome of tobacco plants and translated to synthesize VrPDF1. Recombinant VrPDF1 protein was successfully expressed in seeds of four transgenic tobacco lines (T1-7, T1-8, T1-10, T1-11), among which T1-10 line had the highest recombinant VrPDF1 content, reaching 8.57 g mg-1 total protein. The extract containing recombinant VrPDF1 from the transgenic tobacco lines effectively inhibited the activity of alpha-amylase from the intestine of larvae and weevils. However, the protein extract solution from T1-10 line had the strongest inhibitory effect, so the activity of alpha-amylase was only 18.89% compared to controls. The analysis results of VrPDF1 gene expression in transgenic tobacco plants are fundamental to the transfer of pPhaso-dest-VrPDF1 vector into mungbean to improve bruchid resistance of mungbean and contribute to improving mungbean preservation.


Keyword:     VrPDF1 recombinant protein α-amylase inhibitor transgenic tobacco mungbean weevils.


Citation:

Thao HT, Lan NTN, Tuong HM, Thanh NVT, Son LV, Mau CH. Expression analysis of recombinant Vigna radiata plant defensin 1 protein in transgenic tobacco plants. J App Biol Biotech. 2017; 5 (01): 070-075. DOI: 10.7324/JABB.2017.50112

Copyright: Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license.

HTML Full Text
Reference

1. Chainuvati C, Potan N. and Worasan T. Mungbean and blackgram production and development in Thailand. In: S. Shanmugasun-Daram, and B. T. Mclean (eds.), Mungbean, 1988: 657—668. Proc. 2nd Int. Symp., AVRDC, Shanhua, Taiwan

2. Bui Cong Hien. Warehouses harmful insects. Scientific and technical publishing House, Hanoi, Vietnam; 1995.

3. Chen KC, Lin CY, Kuan CC, Sung HY, Chen CS. A novel defensin encoded by a mungbean cDNA exhibits insecticidal activity against bruchid. J. Agric. Food Chem. 2002; 50: 7258-7263.

4. Chen JJ, Chen GH, Hsu HC, Li SS, Chen CS. Cloning and functional expression of a mungbean defensin VrD1 in Pichia pastoris. J Agric Food Chem. 2004; 52: 2256-2261.

5. Carvalho Ade O and Gomes VM. Plant defensins and defensin-like peptides - biological activities and biotechnological applications. Curr Pharm Des. 2011; 17:4270-4293.

6. Nicole L, Marilyn A. Plant defensins: Common fold, multiple functions. Fungal Biology Reviews. 2013; 26: 121-131.

7. Thevissen K, Ferket KK, Francois IE, Cammue BP. Interactions of antifungal plant defensins with fungal membrane components. Peptides. 2003; 24: 1705-1712

8. Olli S, Kirti PB. Cloning, characterization and antifungal activity of defensin Tfgd1 from Trigonella foenum-graecum L. J Biochem Mol Biol. 2006; 39 (3): 278-83

9. Odintsova TI, Rogozhin EA, Baranov Y, Musolyamov AK, Nasser Y, Egorov TA. Seed defensins of barnyard grass Echinochloa crusgalli (L.). Beauv Biochimie. 2008; 90: 1667-73.

10. Portieles R, Ayra C, Gonzalez E, Gallo A, Rodriguez R, Chacón O, López Y, Rodriguez M, Castillo J, Pujol M, Enriquez G, Borroto C, Trujillo L, Thomma BP, Borrás-Hidalgo O. NmDef02, a novel antimicrobial gene isolated from Nicotiana megalosiphon confers high-level pathogen resistance under greenhouse and field conditions. Plant Biotechnol J. 2010 Aug; 8(6): 678-90. doi: 10.1111/j.1467-7652.2010.00501.x.

11. E.S. Cândido, W.F. Porto, D.S. Amaro, J.C. Viana, S.C. Dias, O.L. Franco. Structural and functional insights into plant bactericidal peptides A. Méndez-Vilas (Ed.), Science against microbial pathogens: communicating current research and technological advances, Formatex. 2011; pp. 951-960

12. Seufi AM, Hafez EE, Galal FH. Identification, phylogenetic analysis and expression profile of an anionic insect defensin gene, with antibacterial activity, from bacterial-challenged cotton leafworm, Spodoptera littoralis. BMC Molecular Biology. 2011; doi:10.1186/1471-2199-12-47.

13. Gan-Hong Chen , Ming-Pin Hsu , Chi-Hsing Tan , Hsien-Yi Sung , C. George Kuo , Ming-Jen Fan , Huei-Mei Chen , Shu Chen , and Ching-San Chen. Cloning and Characterization of a Plant Defensin VaD1 from Azuki Bean. J. Agric. Food Chem.. 2005; 53(4): 982-988

14. Liu YJ, Cheng CS, Lai SM, Hsu MP, Chen CS, Lyu PC. Solution structure of the plant defensin VrD1 from mungbean and its possible role in insecticidal activity against bruchids. Proteins. 2006; 63: 777-786.

15. Wijaya R, Neumann GM, Condron R, Hughes AB, Poly GM. Defense proteins from seed of Cassia fistula include a lipid transfer protein homologue and a protease inhibitory plant defensin. Plant Science. 2000;159:243-55.

16. Schroeder, H. E., Gollasch, S., Moore, A., Tabe, L. M., Craig, S., Hardie, D. C., M. J. Chrispeels, D. Spencer, and TJV. Higgins, T. Bean [alpha]-Amylase Inhibitor Confers Resistance to the Pea Weevil (Bruchus pisorum) in Transgenic Peas (Pisum sativum L.). Plant Physiology. 1995; 107(4), 1233-1239.

17. Maria José De Sousa-majer, Darryl C. Hardie, Neil C. Turner, Thomas J.V. Higgins. Bean α-Amylase Inhibitors in Transgenic Peas Inhibit Development of Pea Weevil Larvae. J Econ Entomol. 2007 Aug; 100(4):1416-22.

18. Swathi AT, Divya K, Jami SK, Kirti PB. Transgenic tobacco and peanut plants expressing a mustard defensin show resistance to fungal pathogens. Plant Cell Rep. 2008; 27: 1777-1786.

19. Karimi M., Inzé D., Depicker A. Gateway vectors for Agrobacterium - mediated plant transformation, Trends Plant Sci. 2002; 7: 193 -195.

20. Topping J.F. Tobacco transformation. Methods of Mol. Biol. 1998; 81: 365 - 372.

21. Edwards K., Johnstone C. and Thompson C. A simple and rapid method for the preparation of genomic plant DNA for PCR analysis. Nucleic Acids Res. 1991; 19: 1349.

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

23. Sun H-J, Cui M, Ma B, Ezura H. Functional expression of the tastemodifying protein, miraculin, in transgenic lettuce. FEBS Lett. 2006 ; 580: 620-626.

24. Bernfeld P. Amylase  and β. Methods Enzymol. 1955; 1:149-154.

25. Hoang TT, Ho TM, Nguyen TTV, Le SV and Chu MH. Vigna radiata plant PDF1 gene (DNA), cultivar Dautam. GenBank: LN901494.1. 2015.

26. Franco OL, Murad AM, Leite JR, Mendes PA, Prates MV, Bloch JC. Identification of a cowpea gamma-thionin with bactericidal activity. FEBS Journal. 2006; 273: 3489-3497.

Article Metrics

217 Absract views 213 PDF Downloads 430 Total views

Related Search

By author names

Citiaion Alert By Google Scholar


Similar Articles