花椰菜ACC氧化酶基因的克隆及其RNAi对内源基因表达的抑制作用

根据亲缘关系较近的几种作物ACC氧化酶氨基酸序列，设计一对简并引物，从花椰菜(Brassica oleracea var．botrytis)基因组中获得长1202bp的候选片段。序列分析表明该基因含有3个外显子(Exon)、2个内含子(Intron)，编码区序列长756bp，编码252个氨基酸。同源性分析发现其与青花菜(Brassica oleracea var．Italica)上已发表mRNA序列同源率为99％(GenBank序列号：X81629)，推断该基因为花椰菜ACC氧化酶基因，命名为BoACO(GenBank登录号：AY676466)。在此基础上，用BP克隆的方法构建BoACO的RNA干涉(RNAi)载体pHBACO，对花椰菜进行遗传转化，获得卡那霉素抗性转化植株5棵，分子检测证实外源片段成功导入其中3棵花椰菜基因组中。Northern杂交分析显示：转基因植株内源ACO基因转录的mRNA被降解。ACC氧化酶活性分析进一步表明，外源基因的导人大大地降低了ACC氧化酶活性。

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.