关键词: 家蚕, 细胞色素氧化酶, CYP18A1基因, 克隆, 序列分析, 转录活性

Abstract: Fundamental events in the life of insects such as growth, development and reproduction are regulated by ecdysteroids (molting hormone), especially during molting and metamorphosis. The available amino acid sequences from GenBank of the orthologous CYP18A1 genes, which are candidates for the ecdysone 26-hydroxylase, was used to BLAST search against the silkworm genomic sequences database, and an ortholog in Bombyx mori was identified. In order to obtain full-length cDNA in B. mori, we used this sequence as a probe and performed in silico cloning based on the B. mori EST database. A contig containing 1 737 bp was assembled on the basis of several ESTs, which extended into 5′-UTR. It was then verified by RT-PCR. PCR product was purified and ligated into pMD18-T vector. The recombinant clones were sequenced, which had the same sequence as the predictive contig. It contains an ORF of 1 623 bp encoding 541 amino acids, termed as B. mori CYP18A1 by the P450 nomenclature committee (GenBank accession number：EF421988). It shares high identities with other orthologs, with its deduced mass 61.67 kD and isoelectric point 8.54. The five conserved motifs of insect P450 enzymes including signature heme-binding region of P450s are present. An alignment of the cDNA sequence with the silkworm genome sequences revealed that there were 6 exons and 5 introns in this gene, and all of them conformed the canonical GT-AG rule. The result of RT-PCR revealed that B. mori CYP18A1 not only showed a temporal and tissue-specific expression profile, but also exhibited a distinct expression pattern which closely coincided with the reported peak of ecdysteroid in the haemolymphs of B. mori. This further suggests orthologous CYP18A1 gene in insects is closely related to ecdysteroid homeostasis.

Key words: Bombyx mori, cytochrome P450, CYP18A1 gene, gene cloning, sequence analysis, transcriptional activity