Vu Thanh Thanh NGUYEN, Van Son LE, Hoang Mau CHU, Thi Xuan Thuy VI, Hoang Duc LE

Expression of the ZmDEF1 gene and α-amylase inhibitory activity of recombinant defensin against maize weevils

Plant defensins are multifunctional small cysteine-rich proteins. They are active against fungi, bacteria, and many viruses, and they inhibit trypsin and α-amylase activities. In this study, we expressed the maize defensin gene (ZmDEF1) in tobacco seeds in order to establish the basis for generating transgenic maize plants resistant to weevils. The ZmDEF1 gene was isolated from Maison, a Vietnamese local maize cultivar, which is well known for having the highest resistance to weevils among other local cultivars. The ZmDEF1 gene was cloned into a binary vector, pBetaPhaso-dest, which carries phaseolin, a seed-specific promoter, to direct defensin expression in tobacco seeds. We obtained 13 transgenic tobacco lines from Agrobacterium-mediated transformation and regeneration, called the T0 generation. T0 s seeds (called the T1 generation) were collected and analyzed for ZmDEF1 gene expression. Reverse-transcription polymerase chain reaction (RT-PCR) results showed that 4 out of 13 lines (T1-1, T1-3, T1-10, and T1-17) expressed the ZmDEF1 gene at the transcriptional level. These lines were further analyzed by real-time RT-PCR until their transcript expression could be identified. The results showed that the line T1-17 expressed it at the highest expression level. The western blot method also showed that ZmDEF1 was expressed in all four lines above. These transgenic seeds can inhibit maize weevils α-amylase activity. Extracted protein from transgenic lines reduced weevil α-amylase activity by 67.9% 71.4% in comparison with protein extracted from nontransgenic plants. These results will hopefully provide a useful background to create transgenic maize plants with high resistance to weevils.

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Baulcombe D (2004). RNA silencing in plants. Nature 431: 356-363.
Bernfeld P (1955). Amylase, α and β. Methods Enzymol 1: 149-154.
Balandin M, Royo J, Gomez E, Muniz LM, Molina A, Hueros G (2005). A protective role for the embryo surrounding region of the maize endosperm, as evidenced by the characterisation of ZmESR-6, a defensin gene specifically expressed in this region. Plant Mol Biol 58: 269-282.
Colilla FJ, Rocher A, Mendez E (1990). γ-Purothionins: amino acid sequence of two polypeptides of a new family of thionins from wheat endosperm. FEBS Lett 270: 191-194.
Edgertom MD (2009). Increasing crop productivity to meet global needs for feed, food and fuel. Plant Physiol 149: 7-13.
Gutierrez-Campos R, Torres-Acosta JA, Saucedo-Arias LJ, GomezLim MA (1999). The use of cysteine proteinase inhibitors to engineer resistance against potyviruses in transgenic tobacco plants. Nat Biotechnol 17: 1223-1226.
Lin KF, Lee TR, Tsai PH, Hsu MP, Chen CS, Lyu PC (2007). Structurebased protein engineering for alpha-amylase inhibitory activity of plant defensin. Proteins 68: 530-540.
Liu YJ, Cheng CS, Lai SM, Hsu MP, Chen CS, Lyu PC (2006). Solution structure of the plant defensin VrD1 from mung bean and its possible role in insecticidal activity against bruchids. Proteins 63: 777-786.
Livak KJ, Schmittgen TD (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2ΔΔC T method.Methods 25: 402-408.
Mahesh BC, Kenneth JB, Timothy CH (2003). Module-specific regulation of the β-phaseolin promoter during embryogenesis. Plant J 33: 853-866.
Melo FR, Rigden DJ, Franco OL, Mello LV, Ary MB, Grosside Sa MF, Grossi DESA, Block CJR (2002). Inhibition of trypsin by cowpea thionin: characterization, molecular modeling, and docking. Proteins 48: 311-319.
Mirouze M, Sels J, Richard O, Czernic P, Loubet S, Jacquier A, François IE, Cammue BP, Lebrun M, Berthomieu P et al (2006). A putative novel role for plant defensins: a defensin from the zinc hyper-accumulating plant, Arabidopsis halleri, confers zinc tolerance. Plant J 47: 329-342.
Murthy PS, Borse BB, Khanum H, Srinivas P (2009). Inhibitory effects of Ajowan (Trachyspermum ammi) ethanolic extract on A. ochraceus growth and ochratoxin production. Turk J Biol 33: 211-217.
Pelegrini PB, Lay FT, Murad AM, Anderson MA, Franco OL (2008). Novel insights on the mechanism of action of α-amylase inhibitors from the plant defensin family. Proteins 73: 719-729.
Pelegrini PB, Franco OL (2005). Plant γ-thionins: novel insights on the mechanism of action of a multifunctional class of defense proteins. Int J Biochem Cell Biol 37: 2239-2253.
Rahmaty R, Khara J (2011). Effects of vesicular arbuscular mycorrhiza Glomus intraradices on photosynthetic pigments, antioxidant enzymes, lipid peroxidation, and chromium accumulation in maize plants treated with chromium. Turk J Biol 35: 51-58.
Selitrennikoff CP (2001) Antifungal proteins. Appl Environ Microbiol 67: 2883-2894.
Stotz HU, Thomson JG, Wang Y (2009). Plant defensins: defense, development and application. Plant Signal Behav 4: 1010-1012. Topping JF (1998). Tobacco transformation. Methods Mol Biol 81: 365-372.
Van der Weerden NL, Anderson MA (2013). Plant defensins: common fold, multiple functions. Fungal Biol Rev 26: 121-131.
Vi TXT, Nguyen VTT, Chu HM (2014). Zea mays mRNA for Defensin Protein (Defensin Gene), Cultivar Maison, GenBank: LN650982. Bethesda, MD, USA: NCBI.
Vi TXT, Ho MT, Le VS, Nguyen VTT, Chu HM (2015). Molecular characteristic of defensin1 gene isolated from some corn samples with difference in resistance levels to weevils. Vietnam Science Technology Review 2: 38-43.
Wilson IW, Kennedy GC, Peacock JW, Dennis ES (2005). Microarray analysis reveals vegetative molecular phenotypes of Arabidopsis flowering-time mutants. Plant Cell Physiol 46: 1190-1201.