家蚕P450基因CYP18A1的克隆、序列分析及转录活性

蜕皮激素对昆虫生长、发育和繁殖有重要调控作用，尤其对蜕皮和变态过程。利用GenBank上登录的蜕皮激素C₂₆羟基化酶候选基因CYP18A1的氨基酸序列对家蚕Bombyx mori全基因组数据库进行BLASTP比对，发现了家蚕直向同源基因(ortholog)，其完全编码序列经RT-PCR检测和克隆、测序验证后，再以此为信息探针检索家蚕表达序列标签(expressed sequence tags，EST)数据库进行拼接延伸，获得了一条包括5′非翻译区在内的长度为1 737 bp的cDNA序列，验证结果也表明与电子克隆序列完全一致(GenBank登录号为EF421988，P450命名委员会将其命名为CYP18A1)。该基因的开放阅读框为1 623 bp，编码541个氨基酸，含有包括P450s特征结构域在内的所有昆虫P450基因的5个保守结构域，其推定的分子量为61.67 kD，等电点为 8.54。将该基因cDNA序列与家蚕基因组序列进行比对，结果表明该基因具有6个外显子，5个内含子，外显子／内含子边界符合经典的GT-AG规则。同源性分析也发现家蚕CYP18A1与其他昆虫的直向同源基因具有较高相似性。用RT-PCR方法对家蚕主要发育变态时期与组织进行检测，显示出该基因的转录表达不仅具有时空特异性，而且在表达时期上与已报道的蚕体内蜕皮激素含量变化有紧密的一致性。该研究进一步证实了CYP18A1基因与昆虫体内蜕皮激素代谢平衡相关联。

Molecular cloning, sequence analysis and transcriptional activity determination of cytochrome P450 gene CYP18A1 in the silkworm, Bombyx mori

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.