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Plant defensins are divided into 18 groups and are multifunctional proteins. The Zea mays defensin 1 (ZmDEF1) gene encodes the defensin 1 protein, which can inhibit alpha-amylase in the insect gut. In this study, the ZmDEF1 gene was transferred into two maize cultivars, LC1 and LVN99, to improve weevil resistance in maize. The recombinant ZmDEF1 protein was assessed for its ability to inhibit alpha-amylase in the gut of the larvae of the maize weevil (Sitophilus zeamais Motsch.). ZmDEF1 was cloned into a pBetaPhaso-dest vector, which harbours phaseolin, a seed-specific promoter, and the Agrobacterium tumefaciens strain C58 harbouring the pBetaPhaso-ZmDEF1 vector was used to transfer the ZmDEF1 gene into two maize cultivars using immature embryos. Transformed calluses were selected on selection media containing kanamycin. The stable integration of the ZmDEF1 transgene into the transgenic maize plant genome was confirmed using Southern blotting. The recombinant ZmDEF1 protein of approximately 10 kDa was expressed in three transgenic maize lines from the LC1 cultivar (C1, C3, and C5) and two transgenic maize lines from the LVN99 cultivar (L1 and L3). The ZmDEF1 transgenic efficiency based on the results of PCR, as well as Southern and Western blotting, was 1.32% and 0.82%, respectively, which depends on the genotypes of LC1 and LVN99. The recombinant ZmDEF1 protein inhibited the alpha-amylase activity of the maize weevil larvae, and its ability to inhibit alpha-amylase is 54.52-63.09% greater than the ZmDEF1 protein extracted from non-transgenic plants.

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.