番茄1──氨基环丙烷羧酸(ACC)合成酶基因的反义RNA──核酶嵌合DNA序列的构建

The Construction of Antisense RNA-Ribozyme Chimeric DNA Sequence of Tomato ACC Synthase Gene

根据番茄ACC合成酶基因（LE—ACC2）DNA序列，以番茄(LycopersiconesculentumMill)果实的总DNA为模板，利用PCR技术扩增得到预期大小的该基因编码区内部分DNA序列，插入到质粒载体pGEM—3zf(＋)的BamHⅠ和HindⅢ位点之间后转化E．coliDH—5α，可选出重组子pRE，经酶切，PCR及DNA序列分析证明克隆成功；将pRE上的目的DNA序列以反义方式构建到我室已合成并克隆的含核酶DNA序列的重组质粒pRⅠ的BamHⅠ和HindⅢ之间，构成含有反义RNA-核酶嵌合DNA序列的重组质粒pREⅠ，经酶切及序列分析，结果与预期一致.

According DNA sequence of Tomato ACC synthase gene(LE-ACC2), and using total DNA offruit of tomato (Lycopersicon esculentum Mill) as template, the expected partial DNA Sequence in codingregion of gene was obtainted by PCR amplification and inserted imto pGEM-3zf(+) digested with BamHⅠ and HindⅢ. then we transformed the system into DH5-α and selected the postive recombinant (pRE).The digestion of enzyme, PCR amplification and sequence of DNA analysis demonstrated that the cloningwas successiful. By the antisense way, the DNA sequence from PRE was combined to PRI between BamHⅠ and HindⅢ to consturct PREI containing antisense RNA-Ribozyme chimeric DNA sequence (pRI wasconstructed in our Lab and contains Ribozyme DNA sequence). The restriction map of recombinants andsequence analysis were indentical to the expected results.