番茄ACO基因的克隆及其RNAi对乙烯产量的抑制

筛选番茄幼苗cDNA文库获得与番茄抗性相关的克隆E11，设计引物，利用RT-PCR方法从受到病原浸染的番茄叶片中获得长1018 bp的候选片段，同源性分析发现该片段与其他作物上已发表的ACO基因序列高度同源：同源率从83%~99%，推断该基因为番茄ACO基因家族的新成员。在此基础上，用BP克隆的方法构建该基因的RNA干涉（RNAi）载体pD311，对番茄进行遗传转化，获得卡那霉素抗性植株27棵，分子检测证实外源片段成功导入其中番茄基因组中。对获得的转基因植株的乙烯生成量测定结果表明，RNAi结构的导入大大抑制了内源ACO基因的表达，从而导致乙烯的生成大大降低。

Cloning of ACO Gene and Inhibition of Ethylene Production with RNAi in tomato

A fragment of 1018 bp of ACO gene cDNA sequence was cloned using RT-PCR with two PCR primers designed according to the sequence of tomato cDNA clone (E11). The result of BLAST showed the sequence presented a very high match with the ACO genes from other plants; the homologue is from 83% to 99%. Using the sequence, an RNA interference (RNAi) transformation vector (pD311) was constructed through the way of BP cloning. The transformation was performed to tomato. 27 regenerated plants with kanamycin resistance were obtained. And the transgene integrated into tomato genome was proved with PCR and Southern blotting. The ethylene respiration amount of the RNAi transgenic tomato plants was measured by gas chromatography. The results showed that ethylene evolution was specifically inhibited in the leaves and fruits of transgenic plants.