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

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.     

特异切割番茄 1-氨基环丙烷羧酸合成酶mRNA的核酶基因序列的合成与克隆

根据番茄1-氨基环丙烷羧酸合成酶(ACC合成酶)基因cDNA序列, 按照锤头状核酶作用模式, 设计合成长度分别为40mer和48mer的一对引物,经体外退火、延伸、连接,插入质粒pGEM-3Zf(+)的KpnI和BamHI位点之间,经菌落原位杂交、双酶切、聚丙烯酰胺凝胶电泳和序列分析证明,人工合成了特异切割ACC合成酶mRNA第667-669位点GUC序列的核酶基因序列。 Abstract:In order to interrupting the ACC synthase activity in tomato,a hammerhead ribozyme targeting to cleave GUC triplet at the position 667~699 of ACC synthase mRNA was established and integrated into KpnI and BanHI site of vector pGEM-3Zf(+).Four positive clones were gotten by in situ hybridization.The recombinatant plasmids were digested with restriction endonuclease KpnI and BanHI.By polyacrylamide gels electrophoresis and sequence analysis,the size and the sequence of one of the digested product was the same as those of what we expected.These results showed that the ribozyme gene sequence has been obtained.