| 摘要: |
| 在分析植物液泡转化酶基因保守区序列的基础上,设计1对PCR引物,以番茄(中蔬四号)叶片总RNA的反转录产物为模板,扩增出长为668 bp的cDNA片段,并克隆入pMD-18 T simple载体。测序结果表明,所获得的片段为番茄液泡转化酶基因TIV1的片段；PCR和酶切分析表明,该片段已成功定向连入到双无载体pBinAR的CaMV 35S启动子和OCS终止子间,构建成反义植物表达载体pBinAR-aTIV1；菌落PCR表明质粒成功转化入农杆菌EHA105；瞬时表达证明该反义片段对番茄叶片液泡转化酶活性具有明显的抑制效果。 |
| 关键词: 番茄 液泡转化酶 反转录PCR 反义表达载体构建 瞬时表达 |
| DOI： |
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| 基金项目:国家自然科学基金项目（30370977）；国家“十五”科技攻关项目（2004 BA 516A09） |
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| Construction and transient expression of vacuolar invertase gene antisense expression vector in tomato leaves |
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| Abstract: |
| Total RNA was isolated from tomato (cv.Zhongshu No.4) leaves by using the specific primers designed according to the sequence of vacuolar invertase gene in GenBank.A fragment with the length of 668 bp was amplified by reverse transcription and polymerase chain reaction and cloned into pMD18-Tsimple vector.Sequence analysis showed the amplified fragment was the TIV1 fragment of the vacuolar invertase gene.The fragment was cut by two restriction enzymes SalⅠand BamHⅠ，and inserted into plant expression vector pBinAR between CaMV 35S promoter and OCS terminator to form antisense plant expression vector pBinAR-aTIV1.The pBinAR-aTIV1 was identified by PCR amplification with TIV1 gene primers, and the direction of connection was identified by double restricted enzymes digestion.The result showed that the expression vector was successfully transferred into agrobacteriumtum EHA105 with method of colony PCR.The activity of vacuolar invertase was decreased by transient expression of antisense fragment in tomato leaves. |
| Key words: tomato,vacuolar invertase,RT-PCR,antisense expression vector construction,transient expression |
