| Oilseed rape(Brassica napus) is a major oil crop and an important source of cooking oil, protein and industrial material in China. Weed influences the yield and quality of oilseed rape greatly, so development of herbicide-resistant varieties has very important significance for oilseed rape production. Bentazone and sulfonylurea are two different kinds of herbicide, and used widely for eliminating latifoliate weeds in rice field. Cytochrome P450gene CYP81A6encodes a kinds of monooxygenase, which can confer resistance to bentazone and sulfonylurea in rice. Arabidopsis thaliana and tobacco introduced with CYP81A6gene are also resistant to these two kinds of herbicide. In this study, rice cytochrome P450gene CYP81A6was introduced into Brassica napus line Jia572using an Agrobacterium-mediated transformation. The obtained bentazone and tribenuron-methyl-resistant oilseed rape lines can be used as germplasms in the transgenic breeding. The main results in this study are as follows:(1)135bastar-resistant plants were obtained through transformation using hypocotyls as explants.37transgenic plants were identified by PCR analysis, with the rate of27.4%.(2) The results of southern hybridization indicated that the target gene was integrated into oilseed rape genome with the copy number of1to4, and6plants harbored only one copy foreign gene.(3) The results of semi-quantitative RT-PCR using β-actin as internal reference gene confirmed that all transgenic lines could express CYP81A6gene, and the levels of expression of different lines were varied. The lines CYP81A6-1and CYP81A6-2had the lowest and highest expression respectively.(4) The Ti plants having4or5leaves were tested for herbicide-resistance under various concentrations of bentazone and tribenuron-methyl. At last, all non-transgenic plants as susceptible controls died under0.24g/L bentazone or1.0μg/ml tribenuron-methyl, when transgenic plants grew normally under1.6g/L bentazone or5.0μg/ml tribenuron-methyl. All transgenic and susceptible plants died when the concentrations of bentazone or tribenuron-methyl reached4.8g/L or10.0g/L respectively. These results indicated that CYP81A6transgenic oilseed rape was resistant to bentazone and tribenuron-methyl, with the corresponding safety concentration ranges of0.24-1.6g/L and1.0-5.0μg/ml respectively. |
