诸葛菜OvCYP86MF基因的克隆及其特性分析

为进一步阐明细胞色素P450基因CYP86MF在诸葛菜发育中的分子机理,本文根据已知同源基因保守序列设计特异引物,利用RT-PCR和RACE技术从诸葛菜中获得一个细胞色素P450基因(OvCYP86MF)全长cDNA序列,该序列全长为1876bp,含有1605bp的完整开放阅读框,可编码534个氨基酸,分子量和等电点分别为61.4kDa和6.90,具有细胞色素P450蛋白的典型特征,即保守结构域FNAGPRLCIG;原核表达显示该基因的融合蛋白在体外可以诱导表达;DANSTAR和Clustal W软件分析表明该基因的全长cDNA序列及其编码氨基酸序列与十字花科物种拟南芥相似性很高,达到80%以上,亲缘关系最近;与CYP86C亚家族成员在氨基酸水平上的相似性均高于50%,因此推断该基因属于CYP86C这个亚家族。Northern杂交分析表明该基因在花蕾中特异表达。

Cloning and Molecular Characterizations Analyses of Cyp450 from Orychophrogmus violaceus (L.)

Abstract：OvCYP86MF, a cytochrome p450 gene, was cloned from Orychophrogmus violaceus (L.) OE Schulz by RT PCR and RACE techniques. The whole cDNA sequence was 1876bp in length with a open reading frame(ORF) of 1605bp. The ORF encoded 534 amino acid residues, which had a typically conserved domain of cytochrome P450 cysteine heme iron ligand signatures (FNAGPRLCIG). The fusion protein was expressed by inducing in vitro. Sequence alignment and Phylogenetic tree analysis showed that OvCYP86MF gene shared more than 80% identities with thoses of Arabidopsis thaliana at the levels of complete cDNA sequences and its deduced amino acid sequences. It also showed over 50% identity to every member of CYP86c family at the level of deduced amino acid sequences. It was inferred that OvCYP86MF belonged to the sub family CYP86C. Northern hybridization showed that the gene was expressed only in floral buds.