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

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

Pages : 98-104

View : 9 | Download : 4

Publication Date : 2017-12-01

Article Type : Research Paper

Abstract :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 insert ignore into journalissuearticles values(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 insert ignore into journalissuearticles values(called the T1 generation); were collected and analyzed for ZmDEF1 gene expression. Reverse-transcription polymerase chain reaction insert ignore into journalissuearticles values(RT-PCR); results showed that 4 out of 13 lines insert ignore into journalissuearticles values(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.
Keywords : Defensin, cysteine, ZmDEF1 gene, Zea mays, Sitophilus zeamais Motsch