Molecular Characterizations And Heterologous Expression Of P450 Genes In The Psocid, Liposcelis Bostrychophila Badonnel (Psocoptera: Liposcelididae)

The psocid, Liposcelis bostrychophila Badonnel (Psocoptera:Liposcelidae), is an important stored-product insect. It is distributed worldwide and is commonly found in various processed and unprocessed dry foods in households, granaries, and warehouses. Outbreaks can lead to significant economic losses and rapid resistance to insecticides resulting in control failure have made L. bostrychophila a pest of interest to researchers globally. However, compared to other agricultural insect pests, stored products pest research (including psocids) was mainly focused on control aspects, while basic research was lacking. P450 enzymes (cytochrome P450 monooxygenases), an important enzyme system in insecticide detoxification, are a complex family of heme-binding enzymes found in most organisms. 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 also play important roles in the adaptation of insects to toxic compounds in their host plants, and are involved in metabolism of almost all commonly used insecticides. Because of the diversity of their substrates, P450-mediated detoxification has the potential to confer cross-resistance to toxins independent of their target site, and thus P450-mediated resistance is now regarded as the most frequent type of metabolism-based insecticide resistance. Therefore, we initiated the study on the molecular characterizations and heterologous expression of P450s in L. bostrychophila. The purpose of the current study was to understand the physiological roles of psocid P450s; to explore the relationship between P450s and insecticide resistance in psocids; to identify P450 genes linked to resistance; and to reveal the molecular mechanisms of insecticide resistance in psocids. The research was supported by the National Natural Sciences Foundation (30871631),Program for New Century Excellent Talents in University (NCET-04-0854) and the Science and Technology Innovation Foundation for Graduate Students (kb2008001).The main results are as follows:1 Housekeeping genes cloning and reference selection1.1 Molecular cloning and sequences analysis of housekeeping genesFour housekeeping genes, Lbβ-Actinl,Lbβ-Actin2, Lba-Tubulin, and LbGapdh, were cloned from L. bostrychophila using the combined techniques of reverse transcriptase-PCR (RT-PCR) with rapid amplification of cDNA ends (RACE). The GenBank accession numbers were FJ196622, FJ447483,FJ595242, and FJ595241,respectively. Molecular characterizations of the putative proteins have been predicted by Protparam.The full-length cDNA of Lbβ-Actinl was a 1772 bp sequence with an open reading frame (ORF) encoding 376 amino acids, while Lbβ-Actin2 was 1350 bp in length containing an ORF encoding 376 amino acids. In addition, the 1565 bp cDNA of Lba-Tubulin had an ORF of 1350 bp encoding 450 amino acids and LbGapdh had an ORF of 333 amino acids. Furthermore, the bioinformatics software such as Scanprosite, PSORT, TMHMM, SignalP 3.0 and ProtFun were used to predict the conserved motif, membrane location, transmembrane structure, signal peptide and potential functions of the putative protein. Phylogenetic trees generated from the nucleotide sequences of their coding regions revealed a relationship that was closer to other insects than to mammals. In addition, their 3-D structure models were constructed by SWISS-MODEL. The housekeeping gene sequences obtained will be helpful to future studies of the psocid L. bostrychophila and related species.1.2 Evaluation and validation of reference genes for gene expression profilingSeveral studies have shown that the reference genes used for the quantification of mRNA expression may be affected by the experimental design or cell type, however, this important aspect is often neglected in gene expression studies of insects. In the current study, the stabilities of five reference gene candidates (Lbβ-Actinl,Lbβ-Actin2, Lba-Tubulin, LbGapdh, and 18S rRNA) were systematically analyzed among the developmental stages and between different strains after deltamethrin induction. The aim of this study was to evaluate housekeeping genes of L. bostrychophila for their suitability as reference genes in quantitative real-time PCR (qPCR) using geNorm and NormFinder, thus provide appropriate reference genes to explore the gene expression patterns of the detoxifying and target enzymes in psocids to clarify the resistance mechanisms at a molecular level. According to the analysis by geNorm, we ranked the stability of the five psocid housekeeping genes after insecticide induction in the following order:Lbβ-Actinl>LbGapdh> Lba-Tubulin>Lbβ-Actin2>Lb18S rRNA. Furthermore, the mRNA expression of selected psocid genes at different life stages was ranked in the following order:Lbβ-Actinl>Lba-Tubulin> Lbβ-Actin2>LbGapdh>Lb18S rRNA.Additionally, the ranking for different strains was Lbβ-Actinl>LbGapdh>Lba-Tubulin>Lbβ-Actin2>18s rRNA. Although there were differences between NormFinder and geNorm, most the results were the same. Therefore, combined the analysis of NormFinder and geNorm, Lbβ-Actinl was selected as the most appropriate reference gene for accurate normalization, while the 18S rRNA is the least stable gene.Two representative reference genes (Lbβ-Actinl,the most stable one; Lb18S rRNA, the least stable one) were applied to determine the expression pattern of a P450 gene encoding CYP6CE2. For deltamethrin induction, the CYP6CE2 transcripts increased significantly normalized to Lbβ-Actinl and Lb18S rRNA, respectively. However, the increase was falsely amplified from 1.9 to 3.4 fold when normalized to Lb18S rRNA.Furthermore, the developmental expression profile of CYP6CE2 normalized to Lbβ-Actinl was different when normalized to Lb18S rRNA, suggesting that the stability of the reference gene had an effect on gene profiling. In addition, the expression stability of Lbβ-Actinl was further investigated in psocids after insecticide induction. These results found that there was no significant difference between the induced and control groups, suggesting that Libβ-actinl was an appropriate internal control for the gene expression profiling in this insect. Our study demonstrated that each candidate reference gene should be validated before use to make sure it is stably expressed under the given experimental manipulation to ensure accurate normalization.2 P450 gene cloning and gene expression profiling2.1 Molecular cloning and sequences analysis of P450 genesFive novel P450 genes, CYP6CE1,CYP6CE2, CYP4CB1,CYP4CC1 and CYP4CD1,were cloned from L. bostrychophila using the combined techniques of reverse transcriptase-PCR (RT-PCR) with rapid amplification of cDNA ends (RACE). GenBank accession numbers were EF421245, EF421246, EU979550, EU979549 and EU979551,respectively. Molecular characterizations of the putative proteins have been predicted by Protparam. The full-length cDNA of CYP6CE1 was a 2025 bp sequence with an open reading frame (ORF) encoded 527 amino acids, while CYP6CE2 was 1822 bp in length containing an ORF encoded 521 amino acids. Furthermore, the 1772 bp cDNA of CYP4CB1 had an ORF encoding 511 amino acids, while CYP4CCl.was 1853 bp with an ORF encoding 504 amino acids. Moreover, CYP4CD1 possessed an ORF of 512 amino acids. Furthermore, the bioinformatics software such as Scanprosite, PSORT, TMHMM, SignalP 3.0 and ProtFun were used to predict the conserved motif, membrane location, transmembrane structure, signal peptide and potential functions of the putative proteins. In addition, the 3-D structure models of CYP6CE1 and CYP6CE2 were constructed by SWISS-MODEL using CYP3A4 as template; however, we were unable to construct structure models for other P450s because the protein database does not currently have a sufficient number of template proteins.2.2 Developmental expression profiles of the five P450 genesIn the current study, we analyzed the developmental expression patterns of the cloned P450 genes from L. bostrychophila to provide insights as to the protein physiological functions. We obtained L.bostrychophila in different, but uniform developmental stages including egg,1st stadium nymph,2nd stadium nymph,3rd stadium nymph,4th stadium nymph, and adult to extract total RNA for gene expression profiling. According to the results of qPCR, transcripts of the five P450 genes were detected at all the life stages tested and none were found to be stage specific. Higher expression levels of CYP6CE2 were observed at nymphal stages than at adulthood. The CYP6CE2 transcripts decrease progressively from early to late developmental stages. The relative quantity of CYP6CE2 was highest in 1st nymph and gradually decreased to the lowest in adults. However, there was no significant difference in expression among the 3rd,4th stadium and the adults (P>0.05). Additionally, as indicated by qPCR, the relative quantity for all life stages of CYP6CE1 was between 1 and 2 folds. The three CYP4 members (CYP4CB1,CYP4CC1 and CYP4CD1)were relatively stable. Similar to CYP6CE1,the relative quantity for all life stages was between 1 and 2 folds. Interestingly, all of them were more highly expressed at adulthood. These results imply that CYP6CE1,CYP4CB1,CYP4CC1 and CYP4CD1 may have important roles in adult psocids, while CYP6CE2 may be more crucial to nymphal psocids.2.3 Expression profiles of the five P450 genes induced by insecticidesWe determined the susceptibility of L. bostrychophila to deltamethrin, paraoxon-methyl and aldicarb using a survivorship bioassay. To determine the induction responses of P450s of the cloned P450s, three insecticides were used to induce the psocids. Based on the bioassay, the psocids were induced by the insecticides in two different regimes:LL, low dose (LC10)+long time (24 h) and HS, high dose (LC50)+short time (0.5 h). Real-time PCR with Lbβ-Actinl as the reference gene was used to determine the relative expression quantity of the P450 genes. As indicated by real-time RT-PCR, four P450 genes (CYP6CE1,CYP6CE2, CYP4CB1 and CYP4CC1)transcripts increased significantly after exposure to 2 mg/L deltamethrin for 24 h (P<0.05), while CYP4CD1 decreased a little. Compared to the control group, CYP6CE1,CYP6CE2, CYP4CB1 and CYP4CC1 transcripts increased 2.8,1.9,1.8 and 2.3 folds, respectively. However, the relative quantity of CYP4CD1 decreased significantly (P<0.05).In addition, we also investigated the time course effects of insecticides (deltamethrin, paraoxon-methyl and aldicarb) on the expression patterns of the five P450 genes following HS regime. With the exception of CYP4CD1,the other P450s expression was highest at 36 h after deltamethrin induction. Compared to the control, CYP6CE1,CYP6CE2, CYP4CB1 and CYP4CC1 transcripts increased 2.5,1.5,3.0 and 1.8 folds. Similar to deltamethrin, paraoxon-methyl showed good induction to CYP6CE1,CYP6CE2, CYP4CB1 and CYP4CC1,but had no obvious effect on CYP4CD1.Twenty four hours after the paraoxon-methyl induction, the relative quantity of CYP6CE1,CYP6CE2, CYP4CB1 and CYP4CC1 reached the 3.9,1.5,6.6 and 2.6 folds, respectively. CYP6CE1 transcripts increased 1.5 fold after 36 h of aldicarb induction, while CYP6CE2 and CYP4CB1 had no significant response. Additionally, the expression of CYP4CC1 was suppressed during the treatment. Interestingly CYP4CD1 transcripts, which had no response to deltamethrin and paraoxon-methyl, increased quickly 8 h after aldicarb induction and reached the peak 36 h later. These results suggest that CYP6CE1,CYP6CE2, CYP4CB1 and CYP4CC1 are inducible to deltamethrin and paraoxon-methyl, while CYP4CD1 was induced by aldicarb. Furthermore, CYP6CE2, CYP4CB1 and CYP4CC1 may be involved in the metabolism of deltamethrin and paraoxon-methyl in L.bostrychophila. Meanwhile, CYP4CD1 was related to aldicarb metabolism.However, we do not know which pathway is involved in controlling P450s transcriptional response to insecticides in psocids. We also do not know what triggers it and how. Further studies including the cloning and analyzing the nuclear receptors and the upstream regions of these P450 genes may provide some information.2.4 Expression profiles of the five P450 genes among different strainsTo verify whether P450s are over-expressed in resistant psocids, real-time PCR with the Lbβ-Actin1 as the reference gene was employed to determine the relative expression quantity of the P450 genes in three different strains of the psocids. Three strains, including susceptible strain (SS), DDVP resistant strain (DDVP-R) and PH3 resistant strain (PH3-R), were started at Southwest University Key Laboratory of Entomology and Pest Control Engineering and subjected to artificial selection to applied pesticides since the 1990s. As indicated by qPCR, four P450 genes (CYP6CE1, CYP6CE2, CYP4CB1 and CYP4CC1)transcripts in DDVP-R and PH3-R strains were significantly higher than SS strain (P<0.05), while the expression of CYP4CD1 was not significantly different (P>0.05), which implied that the overexpressed P450s were possibly involved in psocids resistance. However, the relative expression levels of the overexpressed P450s were relatively low (less than 2 fold). Moreover, as has been demonstrated by our previous studies several other key enzymes such as GSTs and AChE were related to pesticide resistance. Therefore, we assumed that the psocids resistance was a result of the cooperation of the enzymes mentioned above. However, we currently lack sufficient evidence to show that CYP6CE1 and CYP6CE2 are involved in deltamethrin metabolism in psocids. Future studies on substrate specificity of the recombinant proteins will provide direct evidence.3 Heterologous expression of psocids P450 in Escherichia coliHeterologous expression of foreign genes is the core field in functional genomics research, which will provide great insights to the gene function. Thus, biochemical and toxicological properties determination of the recombinant proteins based on the heterologous expression will provide direct evidence to the P450s function study. In the present study, on the basis of Gateway(?) technology, we constructed prokaryotic expression vectors (ligated to pDest 17) for CYP6CE1 and Lb(3-Actinl.Moreover, CYP6CE1 and Lbβ-Actin1 were successfully expressed in E. coli and the recombinant proteins were further confirmed by Western Blot analysis. Additionally, applying the double digestion of BamHI and Xhol restriction enzymes with the DNA recombination technology, we constructed expression vectors for CYP6CE1,CYP4CB1,CYP4CC1 and CYP4CD1 based on pET4317.1a (+)vector. In order to improve the heterologous expression efficiency and convenience to recombinant protein purification, N-terminus modification as suggested by previous researcher was performed for expression vector construction. Signal peptide sequences of the putative protein sequences were discarded, the second codon behind the start codon ATG was changed to GCT. If necessary, four continuous codons of CAT could be added to form a 4×His tag for recombinant protein purification. According to the N-terminus modification, we constructed expression vectors for CYP6CE1,CYP4CB1,CYP4CC1 and CYP4CD1 based on pCW plasmid. Our results formed a solid basis to express psocid P450s effectively in E. coli.4 Polymorphism of CYP6CE1 allelesIt has been reported that mutations of P450 alleles could result in changes in protein structure, substrate recognition and catalytic activity. In this study, three alleles (GenBank accession numbers were EF421245, EU266572 and EU266573) of CYP6CE1 were cloned from 3 strains (DDVP, PH3 resistant strains and susceptible strain) of L.bostrychophila using the combined techniques of reverse transcriptase-PCR and sequenced. According to the sequences alignment, fifteen polymorphism sites were found in the nucleotide sequences of CYP6CElv2 and CYP6CElv3, respectively. We also determined their locations in the sequences. Based on the sequence analysis, the 15 nucleotide polymorphism sites of CYP6CE1v2 resulted in only one amino acid substitution, while five amino acid alterations were found in CYP6CElv3.Protein parameters of CYP6CElvl-3 were predicted by Proparam.As indicated by the prediction, there was no significant difference in properties of CYP6CE1v2 and CYP6CE1v1 protein, which were different from CYP6CE1v3 protein, especially in theoretical pⅠ.Furthermore,3-D structure homology modeling of these three proteins by SWISS-MODEL demonstrated that three amino acid substitutions in CYP6CE1v3 resulted in its structure alteration.In summary, in the present study we cloned the full-length cDNA of four housekeeping genes from L. bostrychophila, which extended sequence information for Psocoptera and provided potential molecular markers to investigate the evolutionary relationship between the Psocoptera and other insects. Five novel P450 genes were isolated form L. bostrychophila, which formed a solid basis to explore the physiological functions undertaken by P450 enzyme system.We systematically evaluated and validated the expression stability of housekeeping genes, and selected only those stably expressed as reference. This constructed a platform for the gene expression profiling in L. bostrychophila using real-time PCR. Based on the reference selection, we determined the developmental expression patterns of these P450s and their expression profiles in the adults after exposure to several insecticides, as well as the relative expression quantity in different strains showing different resistance to DDVP and PH3.Furthermore, we constructed different expression vectors for heterologous expression of psocid P450 in E. coli, in which CYP6CE1 and Lbβ-Actinl were successfully expressed. Our results will not only provide great insights into exploring the functions of the psocid P450s system in development and physiology, but also provide evidence for clarifying the adaptive mechanisms mechanism of psocids to environment. In the meantime, we may enrich and develop the scientific theoretical study system for the resistance mechanism of stored-product insect pests.