Genetic Transformation Of GmGT-2a Gene And CP4-Epsps Gene Into Soybean By Agrobacterium-mediated Transformation

Soybean (Glycine max (L.). Merr) as one of the most important economical crops, is a primary source of nutrients, including vegetable proteins, vegetable oils and so on. Howerer, the soybean planting area in China has kept decreasing in recent years duing to the dramatic impacts of imported transgenic soybeans, which have much higher yield, better quality and lower costs. Therefore, our soybean industry is facing a serious crisis. Normally, the growth and yield of soybean are susceptible to external environment especially to the abiotic factors, such as salinity, drought, frost and other environmental stresses. Traditional breeding methods usally take a very long time and lots of efforts to improve the ability of resistance to external stresses and thus to increase soybean yield. By contrast, using of transgenic technology can create new soybean varieties with better resistance ability in relative higher efficiency and shorter time. The important of this project is that successful creating of new varieties with better resistant ability can expand soybean acreage while not competing with major grain crops for the land,In this study, the plant expression vector pES002-GmGT-2A was firstly constructed, which contains the trihelix transcription factor gene GmGT-2A as well as the glyphosate resistant gene CP4-EPSPS. Three soybean genotypes,’Huachun No.3’、’Zhechun No.3’ and ’0428’, and two transformation systems, i.e. Agrobacterium-mediated cotyledon node system and Agrobacterium-mediated embryo tip system were used in this study. Finally, the key transcription factor GmGT-2A, which can improve resistance ability to abiotic stresses, was introduced into the soybean genome. The main results of this paper are as follows:Construction and functional verification of a plant expression vector. An efficient and easy-to-use plant expression vector plays an important role in the research of gene functions. In this study, the glyphosate resistant gene CP4-EPSPS was cloned and then used to replace the original selective marker Bar gene of plant expression vector pBA002. The new plant expression vector was named as pES002. A trihelix transcription factor gene GmGT-2A was subsequently ligated into pES002and we got pES002-GmGT-2A. This vector was transformed into Arabidopsis to verify the functions of both CP4-EPSPS and GmGT-2A genes. Results showed that the transgenic lines greatly improved the tolerant ability to both glyphosate and high salinity stress, indicating that both the new selective marker gene and the target gene worked well. Thus, this vector was successfully constructed and could be further used in trans-genetic related researches.Agrobacterium-mediated soybean transformation. In this study, three genotypes and two transformation systems as mentioned above were used. Transformation events were confirmed using target gene-specific and selectable marker gene-specific PCR analysis as well as application of glyphosate to seedlings. The results indicated that the two genes have been successfully integrated into the soybean genome.