| The citrus is the second largest fruit tree in China. Zhejiang Province is a main produce area.Facing to the fierce marketing competition, how to improve the quality of citrus fruits and cultivatesuperior varieties have become the key to develop the citrus industry. Currently, the researches on citrusfruit quality mainly concentrated on sugar and acid metabolism, color, storage and preservation, andcarotenoids metabolism, while researches on citrus fruit ripening-related genes are few. Studying onfruit ripening-related gene expression and its regulation are significant for revealing fruit ripeningmolecular mechanism and improving fruit quality by use of biotechnology. The early study has obtaineda specially expressed citrus fruit ripening-related genes CsPEMI-InvI and constructed a dual expressionvector containing the target gene. Taking the wild Micro-Tom as materials, this study tried to optimizethe genetic transformation system resistant to kanamycin and obtained resistant plants with the fruitripening-specific genes CsPEMI-InvI from Citrus. By use of PCR and qRT-PCR technologies, the studydetected the resistant plants and identified that the CsPEMI-InvI gene has integrated into the genome ofMicro-Tom and expressed stably in the plants. The main results were summarized as follows:（1） By selecting the concentrations of kanamycin and cefotaxime sodium, co-culturetime, Agrobacterium concentration and soaked time, the study tried to optimize the genetictransformation system based on kanamycin selection. According to the optimized system,the study transferred the fruit ripening-specific genes CsPEMI-InvI from Citrus intoMicro-Tom tomato.Taking the cotyledon and hypocotyl of10-day Micro-Tom as explantsand making the Agrobacterium concentration OD600=0.5to1.0and the MS dilutedAgrobacterium concentration OD600=0.1, the explants were put into the dilutedAgrobacterium for20min and then after cultured on kanamycin free MS for2days, theexplants were transferred to subculture MS supplemented with100mg.L-1kanamycin and400mg.L-1cefotaxime. Eventually, the study obtained seventeen transgenic plants.（2） Taking the wild Micro-Tom plants as contrast, the study detected the resistant plants with PCRand qRT-PCR technologies. The results showed that CsPMEI-InvI gene has been successfullyintegrated into the genome of Micro-Tom and expressed stably in the plants.（3） Taking wild Micro-Tom plants as contrast, the study detected the activities of pectinesterase and intervase in the leaves of transgenic plants by biochemical methods. The results showed that thepectin methylesterase activity in the leaves of transgenic plants was lower than that of in the wild plantsand showed significant differences. While it showed no obvious difference in acid intervase activity.We verified primarily CsPMEI-InvI gene is pectinesterase inhibitor. |
