Yin-Hua Chen, Jun-Hong Zhang, Bo Ouyang, Han-Xia Li, Zhi-Biao Ye
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引用次数: 0
摘要
利用根据不同植物间ACO氨基酸的一致序列设计的退化引物,从菜花(brassica oleracea Var. botrytis)基因组中扩增出候选ACO基因1202bp的片段。BLAST结果表明,该序列与其他植物的ACO基因具有很高的匹配度;同源性在83% ~ 99%之间。在该序列中鉴定出3个外显子和2个内含子。mRNA的剪接长度为756 nt,编码252个氨基酸。新基因命名为BoACO,已提交GenBank (AY676466)。利用该序列,通过BP克隆的方法构建了RNA干扰(RNAi)转化载体。将其转化为菜花。获得了5株卡那霉素抗性再生植株。并通过PCR和Southern blotting验证了该基因在花椰菜基因组中的整合。Northern印迹分析表明,该基因在转基因植株中的表达下调。ACO酶活性显著降低。
[Cloning of ACC oxidase gene and inhibition of endogenous gene expression with RNAi in cauliflower].
A fragment of 1202 bp of the candidate ACO gene was amplified from the cauliflower (brassica oleracea Var. botrytis) genome using the degenerated primers which were designed according to the consensus sequence of ACO amino acids among various plant species. The result of BLAST showed the sequence presented a very high match with the ACO genes from other plants; the homologue was from 83% to 99%. Three exons and two introns were identified in this sequence. The spliced length of mRNA was 756 nt and encoded 252 amino acids. The putative new gene was denominated BoACO, and submitted to GenBank (AY676466). Using the sequence, we constructed an RNA interference (RNAi) transformation vector through the way of BP cloning. The transformation into cauliflower was performed. Five regenerated plants with kanamycin resistance were obtained. And the transgene integrated into cauliflower genome was proved with PCR and Southern blotting. The expression of this ACO gene is down-regulated based on the Northern blotting in the transgenic plants. The activity of ACO enzyme was depressed significantly.