Genome-wide Structural And Evolutionary Analysis Of Cytochrome P450 Genes, And CDNA Cloning And Function Of The Key Gene CYP18A1 In The Silkworm, Bombyx Mori

Cytochrome P450 enzymes(mixed function oxidases)are a complex family of heme-containing enzymes found in virtually all aerobic organisms,which is one of the oldest and largest gene superfamilies.Cytochrome P450(P450s)evolved from a common ancestor which has existed for more than 3.5 billion years.Many functions are attributed to cytochrome P450s including the metabolism of a wide variety of both endogenous substrates and xenobiotics.The genome of each insect species may carry a hundred or so different P450 genes.They do many things:P450.enzymes are found in the biosynthetic and metabolic pathways of ecdysteroids and juvenile hormones,they also metabolize insecticides,resulting either in bioactivation or,more often,in detoxification,the latter process being enhanced in many strains with metabolic resistence to insecticides.Furthermore, P450 metabolism of certain plant chemicals is often the key to the adaptation of insect herbivores to their host plants.Silkworm(Bombyx raori)is the model organism for Lepidoptera,the second biggest order in insects,which includes the most disruptive agricultural pests.With the development of biotechnology,B.mori has been used as an important bioreactor for the production of recombinant proteins.Lepidopteran genome information not only makes a strong impact on improving sericulture, but also facilitates the development of new pest controls.On basis of the completing genome sequence database of silkworm,coupled with its ESTs and the whole genome microarray,we studied the silkworm cytochrome P450 by analysis of bioinformation and comparative genomics.We also analyzed the structure and evolution of the CYP9A cluster and studied on the cloning and function of orthologous CYP18A1,a candidate for the ecdysone 26-hydroxylase.The main results present as follows.1.Analysis of cytochrome P450s in a completing silkworm genome sequenceThe cytochrome P450s in 24 species available from GenBank or P450 homepage were used to scanne the completing silkworm genome sequence,we identified 84 putative cytochrome P450s in silkworm genome.Among of them,78 appear to be functional P450 genes,six are probable pseudogenes.The genetic map shows that 80 P450s are distributed on 19 chromosomes,eight clusters with over three genes are organized in silkworm genome,with up to 9 member genes in the largest cluster.Of six probable pseudogenes,2 are characterized by deletions,3 by the presence of several stop codons in the open reading frame(ORF)and one separated from their parent loci but never represented a functional gene.Five probable pseudogenes are distributed into the different clusters,maybe they are duplications in the different evolutionary events.This constructs first report on gene clusters and pseudogenes in P450S superfamily of the silkworm.These insect conserved motifs of the protein were used as milestones in the gradual elaboration of full-length P450 sequences.As a result,70 functional P450s with complete open reading frame are identified by Wise2 and EST assembly.All them were named by the P450 Nomenclature Committee,78 functional P450-like genes can be classified into 26 families and 47 subfamilies,and were used to BLAST search against the ESTs database,40 genes have at least one EST,while 44 genes are not represented in the EST collection.Among of them,more P450s from Clan2 and Mito.CYP.clan,especially P450s involved in ecdysteroid biosynthesis,have ESTs as expression evidence than the other P450s from Clan3 and Clan4,which suggest that these P450s from Clan2 and Mito.CYP are essential housekeeping genes which require a high fidelity because of functional constrains.The whole genome microarray also reveals that the different P450s have distinct pattern of temporal and tissue-specific expression.Others unexpressed should be inducible expression.We have drawn the first map of silkworm P450 intron positions and phases compared to the phylogenetic tree.There is a relatively good correlation between intron conservation and phylogenetic relationship between members of the P450 subfamilies.The orthologous CYP15C1 is also identified,which catalyzes epoxidation of methyl famesoate to juvenile hormoneⅢin cockroach corpora allata.2.Comparative genomics analysis of P450s among different insectsThe silkworm and the fruit fly(Drosophila melanogaster),which is the most well-studied model organism for insect,a lepidoptera,a diptera,are complete metamorphosis insects,and they almost contain the same numbers of P450s.We have presumed 10 orthologous gene pairs by phylogenetic analysis,and 5 gene pairs among them are involved in ecdysteroid biosynthesis,which require a high fidelity because of functional constrains,and almost retain stringently as 1:1 orthologs.But P450s in Clan3 and Clan4 are the most numerous among insect P450s,it is difficult to find the distinct orthologous gene pairs.They form different separate species-specific clusters in the phylogenetic tree,which is presumedly related to their habitat and ecological niche.Almost all P450s of silkworm in Clan3 are cluster into one clade with CYP6B found in the metabolism of certain plant chemicals,whereas P450s of housefly in this clan are cluster into a different clade with CYP6A related to metabolic resistence to insecticides.Furthermore,there are distinct number and subfamily difference in P450s of Clan3 and Clan4,We can presume that silkworm may not confer an strong metabolic resistance to xenobiotics,instead,it depend much more on additional selective ability of their varied food sources and environment to cope with the ever-changing environment.By comparative genomics analysis,we find there is conserved arrangement of CYP306A1 and CYP18.41 among the three insects.Together,the tight linkage and conserved intron-exon organization indicate an orthologous relationship.Based on the data.on intron-exon organization of P450s in D.melanogaster and Apis,along with the analysis of P450s in silkworm,we draw comparisons between P450s from different insect P450 Clans.The different P450s contain a large member of unique intron positions and phases,the very few introns of insect P450s conserved across CYP Clan are phase 1 introns,while there is phase 0 conserved across CYP Clan intron.The observed results were not what the "intron-early or intron-late" scenario expected.Fewer introns are conserved from insect CYP genes in Clan3 which appear to share the characteristics of "environment response genes" and are related to functional diversity,while a lot of conserved introns of insect CYP genes from Clan2 and mitochondrial P450 which are involved in essential physiological functions have their counterparts in other insect P450s. As compared with other animal phyla P450s,some intron insertion sites of are conserved,although the three phyla diverged some 700.MAY.The result revealed that the conserved intron-exon structure play a vital part in conservation of gene function.Assuming the genome sizes of B.mori,D.melanogaster and A.mellifera are 432Mb,132Mb and 262Mb,respectively.The intron number per gene and the intron sizes are different,B.mori＞A. rnellifera＞D.melanogaster.There is a tendency that they all increase with genome sizes3.Characterization of multiple CYP9A genes and their putative evolution mechanism All four of CYP9A subfamily members in the silkworm were cloned by RT-PCR and designated as CYP9A19-CYP9A22 by the P450 Nomenclature Committee,respectively.Each of the genes contains an ORF of 1593bp in identical length and encodes a putative polypeptide of 531 amino acids.Both of nucleic acid and amino acid sequences share very high identities to each other. Alignment with the cDNA sequences revealed that these paralogs share an identical genomic structure,each comprising 10 exons and 9 introns of variable sizes.The locations of their introns are absolutely conserved,CYP9A19,CYP9A20 and CYP9A21 form a tandem cluster,whereas CYP9A22 separated from the cluster by four tandem alcohol dehydrogenase-like genes.Phylogenetic relationships and structural comparisons indicated that these paralogs arose as a result of gene duplication events.CYP9A22 with oppositional orientation forms a separate one with the members from different organisms in lepidopteran insect,which is more similar to CYP9A14(63%amino acid identical)from H.armigera than to members of its own,indicating an duplication which occur most early than the other two tandem duplicate events.In contrast,CYP9A19 and CYP9A21 displayed not only a high degree of sequence similarity in the coding region,but also in the 5'-flanking region, which further confirmed that they probably arose from the most recent duplication event.The analysis of the tissue-related expressions show that all CYP9A genes in silkworm selectively expressed in "first line of defense" tissue such as midgut,exoskeleton and subcuticular fat body, which coincides with the most commonly Clan3 function involved in xenobiotics metabolism and resistance.The other tissue-related expressions do not exclude a physiological function towards endogenous substmtes.The differentially expression patterns of four duplicates in silkworm CYP9A subfamily and their phylogenetic relationships also confirm the inference that there is a positive correlation between expression divergence and the age of duplicate genes.4.cDNA cloning and function study on P450 gene CYP18A1 in the silkworm,B.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 development.The orthologous CYP18A1 gene,which are a candidate for the ecdysone 26-hydroxylase,was identified from B.mori.A gene containing 1 737 bp is assembled on the basis of several ESTs,which extended into 5'-UTR.It contains an ORF of 1 623 bp encoding 541 amino acids,termed as B.rnori CYP18A1 by the P450 nomenclature committee(GenBank accession number:EF421988).It shares high identities with other orthologs including Spodoptera littoralis CYP18A1 genes.The result of RT-PCR and the whole genome microarray revealed that B.mori CYP18A1 not only acquired 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.The phylogenetic relationship of genes involved in ecdysteroids shows a close relationship between the silkworm CYP18A1 and the members of the CYP2 family, including some mammalian sterorid-metabolising P450s.Based on the analysis of RT-PCR,we prepared dsRNA and performed RNAi(RNA interference)experiment in the silkworm larva.RNAi mediated knockdown drastically reduced CYP18A1 expression and led to the presence of excessive early mature silkworms and moth.Result revealed that the silkworm CYP18A1 is essential for molting and metamorphosis development.The result futher indicates silkworm CYP18A1 has involved in metabolic pathways of ecdysteroids, which may provide novel targets for exploitation in the development of new pest control strategies.