| The pear'Danshangsu'is one of the famous cultivated varieties,and it's native to Dangshan regions of Anhui Province. But its biggest drawback is susceptible to scab, gave growers bring huge economic losses. Cultivating anti-scab varieties is one basic way to solve this problem. In this sthdy, on the ground of developing shoots regeneration from leaves of Pyrus bretschneideri cv. Dangshansu in vitro. We chose'Dangshansu'as plant material, a resistance-related gene fragment was screened through a Suppression Subtractive Hybridization(SSH) cDNA library of pear'Zaosu'(Pyrus bretschneideri cv. Zaosu) as target gene in Agrobacterium-mediated transformation.We look forward to getting anti-scab varieties. The results as following:1. The recombinant expression vector pGEX-PbzsREMORIN and pGEX- Pbzs25 were transformed into E.coli BL21 cells and was induced by IPTG, and the cells specifically expressed a protein by SDS-PAGE.2. Transient expression of recombinant plasmid pBI221-PbzsREMORIN-GFP and pBI221- Pbzs25-GFP were introduced into onion epidermal cells via particle bombardment for subcellular localization. Subcellular localization assays showed that the PbzsREMORIN protein was present in all the onion epidermal cells,the Pbzs25 protein was present only in the cell membrane.3. The ORF(Open Reading Frame Finder) of PbzsREMORIN was introduced into tobacco NC89 using Agrobacterium-mediated transformation. In order to study this gene disease resistance, all transgenic tobacco plants were tested for resistance to Ralstonia solanacearum by artificial inoculation.The transgenic plants have stronger resistance to Ralstonia solanacearum than those of the control plants. These results showed that PbzsREMORIN play a critical role in disease resistance in pear.4. We studied the effects of different subculture media and plant growth regulators on growing influenced adventitious buds regeneration from leaf were examined. The results showed that the optimal combination for leaf regeneration of pear cultivar 'Dangshansu' was NN69+6-BA 3.0 mg·L-1+IBA0.2 mg·L-1at the beginning of culture. 5. Compared with sugar as carbon sources of regeneration, sorbitol was more appropriate, optimal concentration is more suitable for 10 mg·L-1. The highest regeneration frequency of pear cultivar 'Dangshansu' was 74.03%, and the mean bud number per leaf was 1.01. AgNO3 was more excellent than activated carbon and PVP, 0.5 mg?L-1 is best suitable levels.6. The regenerated shoots were rooted on 1/4MS+ 20 mg·L-1IBA+ 40 mg·L-1 phloroglucinol +6 g·L-1sucrose+6 g·L- 1agar,Dark period 7d.7. Kanamycin was not suitable for screening of genetic antibiotic in transformation of Dangshansu. Neomycin concentration of 50 mg·L -1may suitable for as a selection pressure;. 300 mg / L of antibiotic cef was used as the concentration of effective for selection of bud and inhibition Agrobacterium growth.8. In this experiments, leave of'Danshansu'pear was used as materrial, the contents of this transformation system are as follows:leaves should be infected 5 minutes in Agrobacterium with 0.5 OD600, co-cultivated 2 days. |
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