Molecular Cloning And Sequence Analysis Of CDNAs Of Cytochrome P450 CYP345D3 In Tribolium Castaneum (Herbst)

Cytochrome P450-dependent monooxygenases is a very important enzymatic system,which has been found in all examined living organisms systems.These monooxygenases are able to metabolize a phenomenal number of endogenous and exogenous compounds.It has been regarded as a key object in the biology field because of its diversity in structures and functions.A lot of evidences indicated that insect cytochrome P450 monooxygenase mediated resistance is the main mechanism of the development of insecticide resistance.P450 enzymes bind molecular oxygen and receive electrons from NADPH to introduce an oxygen atom to the substrate.In insects,the diverse functions of P450 enzymes range from the synthesis and degradation of ecdysteroids and juvenile hormones to the metabolism of xenobiotics.P450 enzymes play important roles in adaptation of insects to toxic compounds in their host plants and are involved in metabolism of all commonly used insecticides.To date,various kinds of P450 enzymes have been reported in animals,microorganisms,and plants,and classified into more than 36 families.The first insect P450 gene(CYP6A1)was isolated from an insecticide-resistant strain of housefly,Musca domestica.Subsequently,more and more P450 genes were cloned.Till now,more than 1958 sequences of P450 genes have been registered in the GenBank database.In China,the research about the molecular cloning aspect of P450 genes just started recently,including Helicoverpa zea,Aedes albopictus,Plutella xylostella,Culex pipiens Pallens.The red flour beetle,Tribolium castaneum(Herbst)(Coleoptera:Tenebrionidae),is a cosmopolitan and destructive pest of raw and processed cereal grains.For more than 40 years,this species has shown its ability to develop resistance to insecticides and resistant strains have spread geographically,so that it remains as one of the major pests of stored products.The gene cloning of P450 was investigated in the susceptible strains of Tribolium castaneum (Herbst)and the study was supported by National Natural Science Foundation of China.A novel cDNA clone encoding a novel P450 gene has been isolated from the insecticide-susceptible strain of the red flour beetle,Tribolium castaneum(Herbst)(Coleoptera:Tenebrionidae).The nucleotide sequence of the clone,designated CYP345D3,was determined.The cDNA is 1554 bp in length and contains an open reading frame from base pairs 32 to 1513,encoding a protein of 493 amino acid residues and a predicted molecular weight of 57466 Daltons.The putative protein contains the classic heme-binding sequence motif FXXGXXXCXG(residues 430-439)conserved among all P450 enzymes as well as other characteristic motifs of all cytochrome P450s.Comparison of deduced amino acid sequence with other CYP members shows that the CYP345D3 shares 91% identity with the previously published sequence of CYP345D1 from the T.castaneum genome project and the nucleotide sequence identity between them is less than 80%.After submitted to the P450 nomenclature committee,the gene was named CYP345D3 and the GenBank accession number is EU008544.Phylogenetic analysis of amino acid sequences from members of various P450 families indicated closer phylogenetic relationship of CYP345D3 with CYP6 members from other insects than from mammals and more distant to P450 members from other families.The amino acid sequence is shared numerous similarities with P450 of Tribolium castaneum,CYP345D1(91%, XP₉66774);Tribolium castaneum CYP345D2(62%,XP₉69536);Locusta migratoria CYP6H1 (36%,AF115777),Drosophila melanogaster CYP6G1(36%,NM₁36899);Musca domestica CYP6A1(34%,M25367),Culex pipiens pallens CYP6F1(34%,AY662654);Culex pipiens quinquefasciatus CYP6E1(33%,AB001323),Musca domestica CYP6C1(33%,U09233),CYP6D1 (33%,U15168),Papilio polyxenes CYP6B1(33%,Z29624),Tribolium castaneum CYP346A1(32%, XP₉67901),Rattus norvegicus CYP3A1 01%,NM₁73144),CYP4A1(25%,M57718),Mus musculus CYP1A1(24%,NM₀09992),Tribolium castaneum CYP315A1(24%,XP₉70122).The results proof that the sequence CYP345D3 cloned througn experiment has closer phylogenetic relationship with CYP6 family members of the P450.In addition,the cDNA cloning of P450 complete sequence in T.castaneum has provided some basic information on the molecular mechanism of altered P450 and over-expression.This will contribute to development of molecular diagnostic technique for T.castaneum resistance in field, further understanding of the molecular toxicological characteristics,and paving the way for designing new insecticides and developing new strategies for pest management.