Construction Of Rice Chloroplast Transformation Vector Containing Herbicide Resistance Marker And Molecular Identification

Abstract:Compared with nuclear transformation, chloroplast transformation has a number of unique advantages, such as fixed-point integration, high copy and environmental security. To date, chloroplast transformation technology is relatively mature, especially for dicotyledons, for example tobacco, while in the monocotyledonous plants, the technology is still in its infancy. In order to promote rice chloroplast transformation technology and apply in rice breeding, rice chloroplast transformation system is necessary to be established. In the study, we attempts to find effective selection markers, construct chloroplast expression vectors contained appropriate components, meanwhile, establish the rice chloroplast transformation system.1. Through rice callus sensitivity test, found that, compared with atrazine, the rice is more sensitive to herbicide Basta, glyphosate. So, in rice chloroplast transformation system, they can be used as feasible screening agents.2. Choosing T7g10leader sequence and eight amino acid sequences from N-terminus of T7gene10as the translation control sequences, the fusion fragments between bar gene and sgfp gene as selection marker, we constructed rice chloroplast expression vectors pBS. The vector also contained two homologous fragments, trnI and trnA, the promoter Prrn and the terminator TpsbA. Among them, trnI-trnA came from rice chloroplast genome, Prrn and TpsbA were from tobacco chloroplast genome. The expression of vectors pBS was tested in E.coli, and the obvious fluorescence was observed, Western blot analysis demonstrated that the bar gene and sgfp gene were successfully fused. The vector pBS has been transferred to rice by particle bombardment method. Subsequently, we gained the resistant callus, and green fluorescence was detected by confocal microscopy.3. We investigated the impact of Co60y radiation on transformation efficiency of biolistic method through rice nuclear transformation vector pC1301-egfp. The result indicated that40Gy could significantly improve the transformation efficiency. In the future work, this will lay a foundation for rice chloroplast transformation.