| 'Zhongli 1'pear is a new early-ripening and high-quality cultivar, with a bright marketing prospect. The storage life of'Zhongli 1'fruit is very short, which makes it easy to deteriorate and lose market value during storage period. Therefore, the study on improving storage quality is essential. Ethylene is a type of ripening hormones in plants, and plays an important role in controlling the postharvest ripening and senescent process of pear fruits, so the fundamental way to delay senescent process and prolong shelf life is to inhibit the endogenous ethylene synthesis. l-aminocycloprane-1- carboxylic acid oxidase is the key rate limiting enzyme of ethylene biosynthesis in plants, which converts l-aminocycloprane-1- carboxylic acid (ACC) to be ethylene. We can infer that the content of endogenous ethylene would be reduced if the expression of ACC oxidase gene were blocked. The study was carried out based on this theory and antisense RNA technology. In this study, with'Zhongli 1', adventitious shoots regeneration system and genetic transformation system were established, and furthermore, the plant antisense expression vector of pear ACC oxidase gene was built up and transformed into'Zhongli 1'pear by Agrobacterium LBA4404, and transgenic plants were obtained eventually. The study would provide valuable reference to researches on hindering the expression of ACC oxidase gene in plants for reducing ethylene biosynthesis by genetic engineering. The main results are as follows:1. Effective and stable adventitious shoots regeneration system for'Zhongli 1'pear was establishedTender leaves were excised from 25-30d old subculture plantlets of in vitro growing'Zhongli 1'pear, and three horizontal cuts along the midribs of the leaves were made. Then leaves were inoculated on regeneration medium ( NN69+TDZ 1.5mg/L+IBA 0.5mg/L+agar 6g/L+sucrose 20g/L) with the abaxial surface down. After a continuous darkness culture for 21d, they were cultured under a 14/10 light/dark photoperiod regime. On the average, 85% adventitious shoots regeneration frequency was achieved and 2.72 buds were found on one leaf. Additionally, 1mg/L AgNO3 could increase the regeneration rate.2. Antisense expression vector for'Zhongli 1'pear ACC oxidase gene was constructedAccording to the amino acid sequences of other pear cultivars, a pair of specific primers was designed and employed to amplify'Zhongli 1'pear ACC oxidase gene cDNA sequence. A 520bp length cDNA fragment was obtained by Polymerse Chain Reaction (PCR) from total cDNA of mature'Zhongli 1'fruit. By inversely inserting this fragment into pBI121, antisense expression vector of'Zhongli 1'pear ACC oxidase gene was constructed successfully. Subsequently, the vector plasmid was transformed into Agrobacterium LBA4404, which was then used for gene transformation to pear.3. Genetic transformation system for pear was modifiedTender leaves were pre-treated on regeneration medium (NN69+TDZ 1.5mg/L+IBA 0.5mg/L) with the abaxial surface down. Two days later, they were infected by Agrobacterium with a density of 0.5-0.6 OD600 value for 10min, followed by a 2d-co-cultivation with basal ends upright. Then 200 mg/L Cefotaxime was added into medium to kill Agrobacterium. After another 1 week, leaves were transferred into resistant buds selection medium containing 5mg/L kanamycin. When resistant buds grew up to 1 cm, they were selected by 20mg/L kanamycin.4. Twenty-seven kanamycin resistance plantlets were obtained, and 3 of them were confirmed to be transgenic plantlets by PCR and Southern blot analysis. |
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