The Establishment Of Apple Regeneration System And Functional Characterization Of MxIrt1 Gene

Leave explants from in vitro cultured Malus xiaojineses Cheng et Jiang and Malus baccata (L.) Borkh were used to establish the regeneration system for genetic transformation. The expression of Mxlrtl gene was studied in transgenic tobacco. The results were as follows:1. Under optimum conditions, the regeneration frequency of Malus xiaojineses and Malus baccata' from leave explants are above 90% and 85%. Many factors affecting the regeneration frequency was optimum. It is necessary to put the leaves explants after cutting off in dark for 14~21days. Regenerated shoots were propagated, rooted and then transferred to nutrition water. The differentiation of apple callus is affected by cultivar, explants and cultural condition, in which the cultivar is the most important factor. The adventitious buds are differentiated from calli of Malus xiaojinenses in MS+6-BA 4.0mg/L+NAA1.0mg/L+TDZ4.0mg/L, and the optimum medium for Malus baccata is MS+6-BA6.0mg/L+NAA1.0mg/L+TDZ0.1mg/L. Adventitious buds regeneration from leaves was affected by hormones, dark treatment, explants as well as the orientation of explant placed in the medium.2. Regeneration from leave explants was inhibited at low concentration of kanamycin. 1.0mg/L of Hyg and 250mg/L of Cef didn't affect the regeneration.3. Compared with LBA4404, EHA105 is more efficient for transformation. The optimum coculture time is 2-3d. The optimum bacterial cell density is OD600 0.5.4. Plant expression vector pBIrt, pBIrtR, pBIrt-eGFP and pCWIrt were constructed. PCR and Southern results indicated that Mxlrtl gene was integrated into 16 of 18 regenerated tobaccos. Many speckles and hollows appeared in transformed tobacco leaves and the more roots turned up under iron stress. PCR and Southern blot analysis indicated that Mxlrtl gene was integrated into Malus xiaojinensis genome.5. Mxlrtl gene was expressed in E.coli. The 40kD protein was obtained as expected.6. Southern blot indicated that there are more than one copy of Mxlrtl gene in the genome of Malus Xiaojineses and Malus Baccata. There is 78bp intron in the genomic Mxlrtl gene. Northern blot showed that Mxlrtl gene was expressed in some of the tissues except petal, its expression level is pretty higher in old leaves than young leaves.