Construction Of PBI121-Lyz-GFP Expression Vector And Its Expression In The Callus Of Alfalfa

Taken the different varieties of alfalfa (Gan-nong No.3, M. sativa cv. Xinjiang Daye, Peru) as the objects of study which carrying on the disease-resistant gene for the transformation research of alfalfa. Selected the best culture medium and the combination of hormone to induct the callus; Constructed the expression vector PBI121-Lyz-GFP which contained the GFP and Lyz gene; Optimized the transformation system of alfalfa through the Agrobacterium-mediated and inducted the disease-resistant gene Lyz and GFP into the Hetian alfalfa, the transformation of callus had been detected by the molecular identification and the fluorescent microscope analysis. The result confirmed the Lyz and GFP gene has been integrated into the genome of alfalfa and expressed successfully. The main research results are as follows;1.To induce the callus of alfalfa hypocotyls, the high efficient medium was established by orthogonal design. The data were analysed by visual analysis and variance analysis. The results showed that: MS+2,4-D(2.0mg/L)+6-BA(0.5mg/L)+NAA(1.0mg/L) is the best medium for Peru and Gan-nong No.3 to induce the callus, the effect sequence of three auxins is 6-BA>NAA>KT. MS+2,4-D(2.0mg/L)+6-BA(1.0m g/L)+KT(1.0mg/L)+NAA(1.0mg/L) is the best medium for M. sativa cv. Xinjiang Daye to induce the callus and the effect sequence of three auxins is 6-BA>KT>NAA.2.Construction of plant express vector PBI121-Lyz-GFP including Lyz and GFP gene. GFP gene was inserted into plasmid vector PBI121-Lyz to construct plant express vector PBI121-Lyz-GFP which was selected noting by Kanamycin. Then the new vector was further transferred into LBA4404 by freezethaw method and used for transformation experiment of alalfa.3.Establishment of transformation system: Cultivated 7 days hypocotyls of M. sativa cv. Xinjiang Daye as explants and agrobacterium tumefaciens strain as mediator, the optimized genetic transformation system is as follows: The Kanamycin selective pressure was 40～50mg L^-1; 200 mg·L^-1 Carb could restrained growing of agrobacterium effectively in callus inducing period; the infection time is 10 minutes ; preculture 3 days; 3 days coculture. In a series of replicated experiment, 69 Kanamycin resistant callus of M. sativa cv. Xinjiang Daye were obtained. The observation of GFP expression in the transgenic callus of M. sativa cv. Xinjiang Daye was observed under the fluorescent microscope upon excitation of blue light. PCR amplification and the results of observation showed that the recombined plasmid has been transformed into callus of M. sativa cv. Xinjiang Daye successfully.