Construction of plant expression vectors carrying glnA gene encoding glutamine synthetase and regeneration of transgenic rice plants

摘要

The glnA gene encoding glutamine synthetase (GS) was amplified from Azospirillum brasilense Sp7 with PCR technique. The amplified 1.4-kb DNA fragment flanked with a BamH Ⅰ site at each end was cloned into EcoRV site of Bluescript-SK vector. A recombinant plasmid pGSJ1 containing this 1.4-kb DNA fragment was selected by restriction digestion analysis. The sequencing data also confirmed that the amplified 1.4-kb DNA fragment was undoubtedly the glnA gene of A. brasilense Sp7. Then the 1.4-kb BamH Ⅰ fragment was excised from pGSJ1. A glnA plant expression vector pAGNB92 with rice actin 1 (Act1) promoter was constructed by using colony in situ hybridization to screen positive clones, and 3 rounds of ligation and transformation were performed. Protoplasts isolated from rice (Oryza saliva, L. Japonica) cell suspension line (cv. T986) were transformed with the glnA plant expression vector pAGNB92 carrying neomycin phosphotransferase Ⅱ (NPT Ⅱ) gene by PEG fusion or electroporation. G418~r calli were used to detect NPT Ⅱ enzyme activity. The results show that G418~r calli possess high positive hybridization signal with the frequency of 37%. The regenerated G418~rNPTII~+ rice plants were used for PCR amplification of glnA gene, and a 1.4-kb DNA fragment was amplified from glnA-transgenic rice plants (R0 generation). The results of Southern blot hybridization prove that the 1.4-kb DNA fragment amplified from the total DNA of glnA transgenic rice plants is indeed the glnA gene of A. brasilense Sp7. Northern blot hybridization was carried out using the same glnA gene as probe. The glnA gene was expressed in the transgenic rice plants. Bioassays also confirmed that the glnA transgenic rice plants grew much better than that of the control plants under a condition with nitrogen poor source (0.75 mmol/L).