Cloning And Prokaryotic Expression Of Cytochrome CYP337A1 Gene In The Silkworm, Bombyx Mori

Cytochrome P450-dependent monooxygenases are a very important enzymatic system,which has been found in all living organisms systems examined.P450 enzyme been known as multi-functional oxidase,monooxygenase,fragrant hydrocarbon hydroxy enzyme and medicine supersession enzyme,etc,many kinds of names.These monooxygenases are able to metabolize a phenomenal number of endogenous and exogenous compounds.Cytochrome P450 enzyme department not merely involves a series of endogenous materials such as juvenile hormone(JH)and its analogue,ecdysone,biologic pheromone,But also involve the supersession to exogenous many kinds of compounds such as insecticide,plant secondery materials,environmental pollutant.It has been regarded as a key object in the biology field because of its diversity in structures and functions.To study the molecular characteristics of cytochrome P450 genes from Bombyx mori and further inquire into the relationship between cytochrome P450 and resistance,efforts were given to identify the individual P450 and to express it in heterologous expression systems.The main results are as followed.1)Select the gene sequence.Download many kinds of insect P450s protein sequences such as Drosophila melanogaster, Anopheles gambiae from NCBI.And selected by BLAST P with the predicted protein sequences of the genomic sequences database of Bombyx mori.Get 66 sequences of P450 gene that contain the significant area of ferroheme combined(F_xxG_xxxC_xG),which is a member with characterized heme-binding domain from the CYP3 Clan.Design a pair of primers.Amplify through PCR.Reccive a purpose strip that length is about 1,470 bp.2)Cloning of this gene sequence by the method of T-A cloning. The result deticate that it's ORF(open reading frame)includs a 1,470 bp in length and encodes 489 amino acid residues.The deduced molecular weight is 56.60 KDa that predicted by the Expasy web site.And conform to the rule that the molecular weight of general P450s is 46～60 KDa. Isoelectric point is 8.64.An alignment of the cDNA with the silkworm genomic sequences revealed that there is only one 1,903 bp intron and it conform to the GT-AG rule.The amino acid sequence include 5 characteristic sequences of all insect P450 genes:P450 gene significant sequences that contain the area of ferroheme combined F_xxG_xxxC_xG(428-437);The conservative sequence lie in spiral C that related to the ferroheme combined W_xxxR(116-120);The conservative sequence lie in the position of spiral I that P450 oxygen combines AG_xE/DT(295-299);The totally conservative sequence lie in spiral K that participates steady core-structure E_xxR(353-356);And 'PERF'characteristic sequence P_xxF_xPE/DRT(404-412).According to analysis of protein structure, find that its N end includes the highly dredged signal peptide sequence that,contain 15 dredge water amino acids in all 22 amino acids.3)The homology analyze with other silkworm P450 genes that has already checked.Download the amino acid sequences of 26 cytochrome P450 genes of silkworm that have already checked and landed from NCBI.According to the MEGA 3.1 procedure,use the neighbor joining(NJ)method to set up systematic development tree.The homology relation is near with the gene of CYP6 and CYP9 family that supersed exogenous materials.And it is relatively far with gene of CYP4 family that taking endogenous material of supersession as main fact.The homology relation is farther with the other gene family that metabolize ecdysone and juvenile hormone(JH)in insect.4)The homology analyze with the members of other families.Comparison of deduced amino acid sequence with other CYP members shows that it is more closely related to CYP321A1 from Helicoverpa zea and CYP6P9 from Anopheles funestus,but its overall identity to them is less than 40%.The sequence was assigned to a new CYP family,CYP337A1,by the P450 nomenclature committee.(GenBank land number:EF415297)Download the sequence of highly homology relatived and the representative members of other families.Utilize ClustalX 1.83 to carry on the cluster analysis of amino acid similarly.CYP337A1 have the conservative area of ferroheme combined(PF_xxG_xR_xC_xG)that CYP6 gene family have.But it is sure to take some variations in ETLR and PERF sequence that is highly conservative in CYP6 gene family.It has highest homology relation to the CYP321A1 that has already been diverged from CYP6 family.5)Prokaryotic expressionTo express CYP337A1 protein in E.coli,the ORF of Bmmof was subcloned into the pET50(b) prokaryotic expression vector,the recombinant was transformed into BL21 competent cell,single clone was picked up and incubated in 37℃and the protein is induced by 1 mmol/L IPTG.At the same time,the bacteria transformed with pET50(b)is induced as a control.A obviously bands of the recombinant proteins was found with the apparent molecular weight of 120 KDa.Since the molecular weight of Nus.Tag is 64.3 KDa,while the molecular weight of CYP337A1 was 56.60 KDa respective,the same as prediction.It was indicated that the CYP337A1 gene was expressed in the E.coli.6)optimize the condition of expressionStep forward,use different inducing condition to expression and find that the integrate protein has the biggest espressing amount by the following condition:IPTG density is 0.6 mmol/L,induce time is 2 hours,tempreture is 32℃～37℃.This experiment is the foundation of function analyzing on the protein result of this gene.