Optimization Of The Agrobacterium-mediated Transformation Systems Of Soybean Cotyledonary Node And The Function Evaluation Of Transgenic Soybean Expressing PhyA Gene

Pinxi12, Jidou12, Yudou22are used as plant materials to transfer phyA to the soybean by the method of agrobacterium-mediated transformation system of cotyledon nodes and we have acquired batches of soybean plants. We design some experiments to optimizing the system of transformation of soybean including different cultivars, different lighting conditions and the time of cocultivation by orthogonal design test. The high generation of transgenic plants are used to identify the function of genetically modified soybean.(1) The optimization of genetic transformation system of soybean:in stage of cocultivation, the efficiency of transformation reaches the highest when the variety of soybean is Jidou12, the temperature of cocultivation is24℃, the time of light is10h everyday, the pH of cocultivation medium is5.4and the time of cocultivation is10d following infecting. It can be as the optimization condition of transformation system of soybean in this research area.(2) The mRNA is extracted from T6generation of genetically modified soybean and then cDNA is obtained by reverse transcription on the basis of the self-designed primer of phyA gene. The sequence of cDNA is detected and it turns out that our cDNA shares an99%homology with the target phyA gene(the registration number of GenBank is AF537344.1)by means of BLAST comparison with sequences in GenBank database. This result demonstrates that the target gene can inherit and express stably in transgenic soybean plant.(3) In this study, different lines of genetically modified soybeans with phytase gene are investigated and we assay the ability of secreting phytase in different lines and the ability of uptake and utilization phosphorus in different treatments with phosphorus under nutritional solution culture condition and aseptic medium culture. It turns out that the ability of secreting phytase in root system of high-generation transgenic plants is higher than in the normal plants. The capacity of phytase secreted from roots of different transgenic lines of T6generations of the Jilin35such as T6-12, T6-44, T6-66is2.9-3.5times higher than the control under nutritional solution culture condition, while T6-3, T6-12, T6-44is2.4-2.8times higher than the control under aseptic medium culture. On the condition of dealing with phytic acid phosphorus, phosphorus content of transgenic lines is1.11-3.18times more than the control, especially T6-12and T6-44is2.34and3.18times separately. The characters of transformed plants is superior to the control under the phytate phosphorus processing in sand culture and the single grain yield has increased significantly. The grains per plant of Strain9and Strain13is2.9and3.5times higher than the control and the single grain yield of them is2.2and1.8times separately. It is proved that phytase gene has bean shifted to the soybean genomes and express successfully. Beside, increasing phytase secretion could enhance utilization of organic phosphorus in transgenic plant.