卡特兰ACO基因克隆与反义表达载体的构建

CLONING OF ACC OXIDASE GENE FROM Cattleya FLOWER AND CONSTRUCTION OF ITS PLANT ANTISENSE EXPRESSION VECTOR

以卡特兰(Cattleya)花瓣为试材,提取其总RNA,并根据其他兰花的ACC氧化酶(1-aminocyclopropane-1-carboxylic acid oxidase,ACO)基因保守序列设计了一对特异性引物,通过RT-PCR法克隆得到1条967bp的卡特兰ACOcDNA片断,共编码321个氨基酸残基。序列分析结果显示该克隆片断与已发表的其他兰花的ACO基因序列同源性很高,均在85%以上,尤其与原生种和其近亲属的同源性在95%以上。将克隆的卡特兰ACO片段反向连接到植物表达载体pBI121中CaMV35S启动子的下游,构建了卡特兰ACO基因的反义表达载体pBI121ACC,为进一步应用反义技术培养长花期卡特兰新品种奠定了基础,也首次为应用生物技术延长卡特兰花期做出了尝试。

Total RNA was extracted from flower of cattleya, according to the conserved acid sequence for ACC oxidase in other orchid, we designed a pair of oligo nucleotide primers. Using RT-PCR method, a eDNA fragment about 967 base pair, which encoded 321 predicted amino acid residues, was amplified. The results of BLAST showed the sequence presented a very high match with the ACO genes from other orchid, the homologue was higher than 85 %, and was higher than 95 % with the original species and relatives. An antisense expression vector of the cattleya-ACO gene, which named pBI121ACC, was constructed, and the antisense sequence was controlled by the CaMV 35S promoter. The work laid the foundations of future transgenie research for prolonging the flowering period of the transgenie Cattleya via antisense technology.