家蚕黄酮合酶I基因BmFNS I的克隆与干涉载体的构建

摘要

黄酮类化合物广泛存在于动植物中,具有多种生理活性,黄酮合酶I基因可以催化多种黄酮类化合物的生物合成.根据NCBI已登录的其他物种黄酮合酶I基因的氨基酸序列,与家蚕基因组和EST数据库进行电子克隆获得家蚕的同源基因BmFNS I.克隆测序结果表明该基因长1 451 bp,7个外显子.根据基因序列推测其编码蛋白由345个氨基酸构成,179～295间氨基酸为一2-氧戊二酸和Fe(II)依赖性的加氧酶家族成员的结构域.RT-PCR检测该基因在本试验所调查的家蚕中肠有高表达.最后将该基因片段亚克隆至具有2个反向T7启动子可以用于体外诱导合成dsRNA的L4440载体.%Flavone is widely existing in animals and plants. They have various physiological activity and the synthesiza-tion of them can be catalyzed by flavone synthase I gene. Using the reported amino acid sequences of other organisms and the scaffold and EST sequences of silkworm, silkworm flavone synthase I gene, BmFNS I, was obtained by electronic clone. The gene is 1 451 bp in length and 7 exons in it by cloning and sequencing. The deduced protein contains 345 ami no acids and a structural domain of 2-oxoglutarate (20G) and Fe( II)-dependent oxygenase superfamily located in amino acids 179 and 295. It is shown by RT-PCR experiment that the gene expressed mostly in midgut in all the tissues tested. Then gene was subcloned to L4440,which has two T7 polymerase promoter in opposite orientation to synthesize dsRNA in vitro.