The Study Of KP4Gene Genetic Transformation Of Sugarcane

Sugarcane(Saccharum officinarum L.) is not only the main sugar resource in the world, but it is also the important economic crop in the subtropical and tropical regions. The production of sugarcane has been largely affected by intense insect pests, among which smut is one of the most serious ones. Sugarcane has long growth cycle and lack still is the smut resistant varieties. When the traditional pest control methods are not effective transgenic breeding becomes an alternative approach. In the study, we selected the gene KP4in the corn and wheat which had been proved to be the smut resistant and then conducted agrobacterium-mediated transformation in cultivated variety of sugarcane ROC22with the purpose of improving the sugarcane’s resistance to smut. The main conclusions were:1. The gene KP4plant expression vector pAKP4was constructed with gene bar being the selective marker gene and Actl being the promoter.2. pAKP4was transformed into the embryogenic callus of sugarcane using agrobacterium-mediated transformation.56KP4transgenic plants were obtained by PPT resistance selection and PCR detection.3.7KP4transgenic plants with PCR detection being positive were selected to be detected using southern blotting and5plants showed positive. All the5plants were then detected using RT-PCR and they all showed positive, thus proving the success of gene KP4integration into sugarcane genome and expression of gene KP4at the transcriptional level.4. We extracted the crude protein in the5plants with RT-PCR detection being positive and then evaluated the effects of the crude protein on germination of U.scitaminea Syd. Spores. We found that the crude protein significantly inhibited germination of U.scitaminea Syd. spores. We also infected stem buds of the5plants and calculated the morbidity after field planting. One plant showed resistance (R) to infection. Three plants showed moderate resistance (Ⅰ) to infection. One plant showed susceptible to infection (S). The results indicated that the KP4gene in sugarcane could express protein with activity and improved the sugarcane’s resistance to U.scitaminea Syd.5. The gene KP4prokaryotic expression vector pET-KP4was constructed. The amount of protein KP4expression was the largest after being inducted8hours when the concentration of IPTG was1.0mmol/L and culture temperature was37℃. The protein KP4could satisfy the requirements of polyclonal antibody after purification. Consequently, it constructed a basis for polyclonal antibody preparation and protein KP4expression detection in future.