Construction Of A Vector With Herbicide Resistant Gene And Its Transformation In Rice

Rice is the staple food in China and in the world. In China, rice ranks number one among all the grain crops in growing area, total yield, and yield per unit area. Every year, as a direct result of weed, the rice yield loss amounts to over 10 million tons. However, the control of weeds in rice fields entails lots of manpower, material and financial resources, as well as spraying large amount of herbicide, causing serious environmental pollution.Generation and development of transgenic technology made it possible to create ground-breaking germplasm resources. With the application of plant genetic engineering, herbicide-resistant and high-yield rice varieties are cultivated, which will be economical and effective in controlling weeds in rice fields, thus promoting China's grain production.aorA gene, encoding the 5-enolpyruvylshikimate-3-acetone shikimic acid phosphate synthase (EPSPS), provides resistance to the herbicide glyphosate and the corresponding herbicide is well known for its high effectiveness, low toxicity, no residue in organisms and soil and environmental friendliness.In this study, herbicide resistant gene aroA was constructed on the plant expression vector, and was transformed into different rice varieties by Agrobacterium tumefaciens. Transgenic plants with herbicide resistance was obtained. Meanwhile, issues concerning the procedure of rice transgene were discussed.The research results are as follows:1. With vector pCambial 301 and herbicide glyphosate-resistant gene aroA, plant expression vector pCambia 1301-aroA, with hygromycin-resistant gene (hpt) deleted from the vector, was constructed.2. With the constructed vector, gene aroA was transferred into rice varieties by Agrobacterium-mediated transformation. As a result, totally 16 regenerated plants were obtained, 4 of which were Zhonghua 15 and 12 of which were Shuhui 527.3. PCR assay was carried out for the 16 regenerated rice plant, and 9 showed positive, 8 of which were Shuhui 527 and 1 of which was Zhonghua 15. Finally, 2 of the 9 PCR-positive plants showed herbicide resistance when selected for field test. The result suggested that the target gene had integrated into the genome of the regenerated rice plants, and had fully expressed, resulting in herbicide resistance in the transgenic rice plants.4. Calli derived from mature and immature embryos of 4 different materials was induced. The result showed that callus inducement ratio varied among different genotypes of rice (japonica rice had higher ratio of inducement than indica one), and so did different materials of the same genotype. Calli derived from immature embryos were of better quality and had faster growing rate, but were more difficult to manipulate and had higher pollution rate, when compared with the calli derived from mature embryos.5. Calli of different receptor material was inoculated on medium with different concentration of glyphosate in order to carry out the sensitivity experiment. The results showed that the rice calli had relatively poor sensitivity to glyphosate and needed relatively high selection concentration. Meanwhile, the selection concentration for japonica rice (1000mg/L) is slightly higher than that for indica rice (800mg/L).