Study On Tobacc And Alfalfa Transformation Of AcdS Gene Mediated By Agrobacterium Tumefaciens

ACC deaminase can catalyzes metabolism of ACC to ammonoa anα-ketobutyrate.The gene transformation acceptors selected in this experiment are tobacco NC89 and the Alfalfa cultivar Medicago sativa L."Zhongmu NO.3. The acdS gene, which had been cloned from Pseudomonas putida UW4 and ligated to CaMV35S-2 and rolD promoter, was transformed into tobacco NC89 by Agrobacterium -mediated. In the process of transferring alfalfa by leaf disks, we optimized high -frequency regeneration system. Based on that, gene acdS, was also transformed into Medicago sativa L."Zhongmu NO.3"by Agrobacterium-mediated. The transgenic alfalfa plants were gained and the expression elements could be integrated into the genome of alfalfa by identification.The main results were as following: Transformation of acdS and identification of transgenic tobacco. By PCR, RT-PCR and salt-tolerance analysis, the results indicated that acdS gene had already inserted into the genome in the plants and transcripted to mRNA. The transgenic tobacco palnts of the gene acdS under the transcriptional control of the rolD promoter had stronger salt-tolerance than the other; Select the cotyledon as the explant, conduct the tissue culture systematic research and seek a high differentiation rate explant and the basic culture medium that suitable for this variety. It was indicated that UM +KT 1.5mgL-1 medium had a better effect on embryo induction;Establishment and optimization of genetic transformation system by Agrobacterium-mediated.The optimized genetic transformation procedure was: The edges of cotyledons from aseptic plants which had been cultured for 5~7 days were cut, pre-culture for 2 days then moved to a suspension of Agrobacterium cells. Cell density was adjusted to fall 0.5 at OD600. After 10min inoculation and co-culture 3 days, the explants were transferred to UM containing 500 mgL-1 cefotaxime for a week.Then they were transferred to UM containing 300mgL-1 cefotaxime and 50mgL-1 kanamycin for induction of Kan- resistance callus; Transformation of acdS and identification of transgenic plants.The acdS gene was transformed into Medicago sativa L."Zhongmu NO.3"according to transformation procedure showed above. Kan-resistance callus which derived from explants were transferred to UM containing 1.5mgL-1 kinetin, 100 mgL-1 cefotaxime and 50mgL-1 Kanamycin for embryo induction and development. Embryos derived from callus were transferred to 1/2MS for the formation of roots. Finally, molecular identification of regeneration plants was carried out; the results of PCR and southern Blot identification of acdS gene showed that some alfalfa plants had inserted into the genome.In addition, the results of RT-PCR and cDNA sequencing showed that the gene of acdS had transcribed into mRNA.