番茄LE-ACC_2合酶基因5′端前导序列分离方法的研究

Isolating Method of 5'-End Lead Sequence of LE-ACC_2 Synthase Gene

通过ＰＣ／Ｇｅｎｅ软件对 Ｒｏｔｔｍａｎｎ等人发表的番茄 ＬＥ－ＡＣＣ２合酶的 ５’端 ２．０ Ｋｂ的前导序列作分析，设计了４对特异引物；以番茄果实、叶片的总ＤＮＡ为模板，采用特异ＰＣＲ扩增技术获得了 ２．０， １．８７， １．５８， １．２８ Ｋｂ ４个特异片段，用 Ｔ－Ｖｅｃｔｏｒ技术构建了１个克隆（２．０ Ｋｂ）、３个亚克隆（１．８７，１．５８，１．２８ Ｋｂ），对４个克隆产物进行了 ＤＮＡ序列测定，借助 ＰＣ／Ｇｅｎｅ软件对所获得的各克隆序列进行综合处理与分析，获得了番茄 ＬＥ－ＡＣＣ２ ５’端前导序列的同源率为９９．９％，但第－９７９位的 Ｃ和第－１０７６位的 Ｔ分别为 Ｔ和 Ｃ所替代。对利用 ＰＣＲ技术分离大片段ＤＮＡ的各环节作了较详细的研究。

End Lead Sequence of 2.0 kb DNA of LE-ACC2 synthase gene was isolated using PCR technique. Specific primers were designed as the sequence published by Rottmann W. H., et al. DNA fragments, 2.0, 1.87, 1.58, 1.28 kb in length, were amplified from total genomic DNA of tomato leaf and fruit, which were constructed into one clone (T-vector+2.0 kb) and three subclones (T-vector+l.87 kb/1.58 kb/1.28 kb). A 2.0 kb 5'end lead sequence of LE-ACC2 isolated was fully sequenced. Its homology rate was 99.9 % as compared with the sequence published by Rottmann W H, with -979th C and-1076th T changed into T and C according to PC/Gene software assays.