甜瓜果实酸性转化酶基因反义表达载体的构建(英文)

Construction and study of a vector for antisense expression of acid invertase gene from melon

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。

Improvement of fruit quality is an important object in melon breeding. Application of antisense RNA strategy to reduce acid invertase activity provides a new feasible method for breeding melon cuhivars with high sucrose accumulation. In a previous study we cloned a eDNA fragment of acid invertase from melon fruit into pMD18-T. For constructing antisense melon acid invertase gene vector （Anti-MAI1）, the gene was digested with BamHⅠ and Hinc Ⅱ to release a fragment with 1038 bp coding region,which was then inserted between the cloning sites of BamHⅠ and SmaⅠ of pROK2. Anti-MAI1 was transferred into tobacco mediated by Agrobacterium tumefacions LBA4404. PCR and PCR-Southern blot were performed, and the results showed that MAII had been transformed into the genomic DNA of tobacco.