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筛选番茄(Lycopersicon esculentum)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方法从受到病原侵染的番茄叶片中获得长1018bp的候选片段,同源性分析发现该片段与其它作物上发表的ACO基因序列高度同源,同源率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。
The cDNA library of Lycopersicon esculentum seedlings was screened to obtain the clone E11 related to tomato resistance. The primers were designed and the 1018bp candidate fragment was obtained from the pathogen-infected tomato leaves by RT-PCR. Homology analysis showed that the fragment It is highly homologous to the ACO gene sequence published in other crops with the homology of 83% -99%. It is concluded that this gene is a new member of the ACO gene family in tomato. Based on this, BP RNA interference (RNAi) vector pD311 was constructed by BP cloning method, and 27 kanamycin-resistant plants were obtained by genetic transformation. The molecular analysis confirmed that the foreign DNA fragment was successfully introduced into tomato genome . The obtained ethylene production of transgenic plants showed that the introduction of RNAi structure greatly inhibited the expression of endogenous ACO gene and resulted in greatly reduced ethylene production.