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应用RNA反义技术抑制甜瓜果实发育过程中酸性转化酶活性,促进蔗糖积累,从而为培育优质品种提供了可行的新方法。将已克隆到pMD18-T载体上的甜瓜酸性转化酶基因用BamHⅠ和HincⅡ双酶切,得到该基因编码区1038bp的cDNA片段,将其定向插入到植物表达载体pROK2的BamHⅠ/SmaⅠ克隆位点,构建了甜瓜酸性转化酶cDNA反义表达载体(Anti-MAI1)。采用冻融法将其转入根癌农杆菌LBA4404,得到了完整的Ti质粒表达载体系统。利用叶盘法转化烟草,经PCR和PCR-Southern杂交检测,证明此基因已整合入烟草的核基因组中。
The application of RNA antisense technology can inhibit the activity of acid invertase and promote the accumulation of sucrose during the fruit development of melon, thus providing a feasible new method for cultivating high quality varieties. The melon acid invertase gene cloned into pMD18-T vector was double-digested with BamHⅠ and HincⅡ to obtain a 1038bp cDNA fragment of the coding region of the gene. The fragment was inserted into the BamHI / SmaI cloning site of plant expression vector pROK2, The antisense expression vector of melon acid invertase (Anti-MAI1) was constructed. The recombinant plasmids were transformed into Agrobacterium tumefaciens LBA4404 by freeze-thaw method, and a complete Ti plasmid expression vector system was obtained. Tobacco was transformed by leaf disc method. The result of PCR and PCR-Southern hybridization showed that the gene was integrated into the nuclear genome of tobacco.