| As a low-toxin, broad-spectrum, high efficient herbicide, atrazine is widely used on essentially all annual and perennial plants. Just because atrazine is non-selective, it kills both weeds and rice plants and causes unnecessary loss to peasant. Accordingly, there is a need to produce transgenic rice plants that are resistant to atrazine.In this study, overlapping extensions polymerase chain reaction (PCR) was used to change atrazine chlorohydrolase trzN two bases sites, without changing of amino acid sequence:the BamHI sites GGATCC were changed to GTATCC sites and the KpnI sites GGTACC were changed to AGTACC sites. At the same time the BamHI and KpnI sites were added to the5’end and3’ends.The modified trzN gene fragment and plant expression vector pVec-8Ubi were digested by KpnI and BamHI to contruct the trzN over-expressing vector. The target gene was successfully introduced into rice by Agrobacterium-mediated transformation.54trangenic plants obtained through Hyg selection, and all of them were PCR positive. Three positive plants were analysed by RT-PCR and the results showed that the trzN gene was integrated into rice.Sprouted seedings of wild type rice (Oryza sativa L. cv. Nipponbare) were treated by water, lg/L adjuvant,0.0004g/L,0.004g/L,0.04g/L,0.4g/L and4g/L atrazine. When treated concentration lower than0.0004g/L, rice leaves exhibited no obvious different from control. However, rice leaves withered when they were exposed on0.04g/L atrazine or higher. Atrazine resistance analysis was also carried out between transgenic plants and control:Transgenic seeds were selected by hygromycin for one week, then all of wild type plants and transgenic ones were treated with water,1g/L adjuvant,0.004g/L,0.04g/L and0.4g/L atrazine. Two weeks later, transgenic plants showed higher survival rate than wide-type ones. Meanwhile, the transgenic rice seeds were selected by homomycin, then50mg/L atrazine was sprayed on leaves of rice plants in the period of4leaves for two weeks. After four weeks, transgenic plants showed atrazine-resistance and growed higher than untransformred control. |
