The Study On Transformation And Expression Of Grobacteria-mediated Lyz-GFP Genes In Creeping Bentgrass

Agrostis stolonifera.L, Agrostis, grass family, is a kind of thin herb and the most important cold season type lawn throughout the world, and also the most widely used grass in green of golf and bowling. Because of its strong resistance ability in cold, drought, shadow and low-grade clip, the bent grass is currently widely used in landscaping. But the bent grass is weak in disease resistance, and is susceptible to vermin, especially to fungi disease, which bring a great lose in turf production and operation. Based on this situation, agrobacterium mediated approach was used to transfer the spectral disease resistance genes that carry fluorescent marks --- the isozyme gene in to bent grass, to let them get expressed and increase the disease resistance ability.In the study, the regeneration ability of the 2 bent grass varieties Penn A-1 and Penn A-4 were further compared, and were established successfully the regeneration system of the 2 varieties, compared the regeneration effect, and were finally conducted the gene transformation study to obtain the genetically modified plant with pBI121-Lyz-GFP gene successfully. Furthermore, fluorescence detection and PCR amplification test were conducted and obtained the existing proof of the target genes in transformational Penn A-1.The contents are listed as follows:1. The regeneration capability of bent grass Penn A-1 and Penn A-2 were compared, and excellent plant regeneration system was successfully established.The mature seeds of bent grass were used as explants, and were studied the callus induction, differentiation and root culture. The results indicated that during callus induction, 2mg/L 2,4-D +0.1 mg/L 6-BA and 4mg/L 2,4-D +0.1 mg/L 6-BA were the best combinations on MS media for Penn A-1 and Penn A-4, respectively, and the induction rates were 36% and 82.4%, respectively. MSO medium was found performed best in the differentiation period of callus, and the calli differentiation rates were 42.5% and 44.5%, respectively. According to comparison, the regeneration ability of Penn A-4 was found better than Penn A-1.2. The transfer conditions were studied, and genetic transformation system was optimized, and the transformational plants of Penn A-1 and Penn A-4 were obtained.21d was used as one subculture period, and the embryonic calli of Penn A-1 and Penn A-4 which had been continuously subcultured for 3 to 5 times were used as explants, and were infected with agrobacterium LBA4404 that carried with pBI121-Lyz-GFP to make the double gene Lyz-GEP get introduced into Penn A-1 and Penn A-4. The main factors influencing the gene transformation of bent grass were studied and the corresponding values were ascertained: the best carbenicillin concentration was 300mg/L, the most appropriate infection time was 10 to 15min, the optimum OD600 values of bacteria liquid concentration were 0.3 to 0.5 for Penn A-1 and 0.5 to 0.9 for Penn A-4, the effective integration and transformation of genes could be done after 3d of coculture.3. According to florescence detection and PCR amplification, the positive transgenic plant Penn A-1 was obtained.