| Liposcelis entomophila(Enderlein)(Psocoptera: Liposcelididae), an important argriculture insect harming stored-products, has turned out to be a kind of serious pest insects of stored commodities in worldwide, especially in China. Recently, due to psocids’ impact on economic of stored grains, contamination of food, and their role in the transmission of harmful microorganisms, they are becoming a new risk for global food security and safety. An apparent insecticides resistance have been observed in a number of early control failures with psocids. Compared to other stored pests, the studies of molecular mechanisms were lacking. P450 enzymes(cytochrome P450 monooxygenases), an important enzyme system in insecticide detoxification, could be found in most organisms. The P450 s are known to play significant roles in the metabolism of insecticides and are involved in metabolism of a lot of exogenous such as drugs, pesticides, plant toxins, chemical carcinogens and mutagens, as well as endogenous compounds such as hormones, fatty acids, and steroids. Therefore, our study facilitates the understanding of the response pattern of P450 s in L. entomophila under the stress of xenobiotics. Firstly, we generated a c DNA library of L. entomophila and identified a lot of detoxification enzyme genes. Secondly, based on the transcriptome data, we cloned five full-length P450 genes. Thirdly, the expression pattern of P450 genes under different chemical exposure was examined. Developmental stage-specific and both sexes expression patterns of CYP P450 genes were also examined. In additon, part of the eukaryotic expression system of P450 in L. entomophila was established preliminary. Finally, the comparison of P450 s activities in sensitive and resistant population from L. entomophila was conducted. Our results provided important information to the functional verification of cytochrome P450 system. The main results are as follows:1 Transcriptome analysis of L. entomophila and identification of detoxification enzymes genesIn order to obtain a L. entomophila comprehensive transcriptome, the c DNA library was generated by Illumina HiseqTM2000. A total of 54,220 Unigenes were identified. Using BLASTX with a cut-off E value of 10-5, there were 33,404 Unigenes successfully annotated in Nr, 25,637 in Swiss-Prot, 23,068 in KEGG, 11,773 in COG and 19,355 in GO databases, respectively. To annotate L. entomophila unigenes, all ofthe assembled sequences were subjected to BLASTx similarity search against the Nr database to predict their putative functions. According to the best hit in the Nr database, more than half of annotated unigenes(20,017 unigenes, 59.93%) had strong homology with human body louse(Pediculus humanus). The result of GO classification showed that 19,355 Unigenes were divided into three categories and 61 functional groups. And a total of 11,773 Unigenes were classified into 25 COG families.A large number of genes potentially involved in insecticide resistance were manually curated. There were 68 putative cytochrome P450 genes with an average length of 1,124 bp identified from the Nr annotation. Among these P450-related unique transcripts, 35, 18, 9 and 6 Unigenes were divided to CYP3, CYP4, CYP2 and mitochondrial CYP family, respectively. We also identified 37 GSTs Unigenes with an average length of 551 bp, which were assigned to the Delta, Omega, Sigma, Theta, Zeta and Microsomal families. A total of 19 sequences encoding CCEs with an average length of 1,427 bp were identified from the Nr annotation results among which were divided into Clade A, Clade D, Clade E, Clade F, Clade H and Clade J, respectively.2 c DNA cloning and c DNA analysis of P450Five CYP P450 genes with complete ORFs were validated and submitted to the CYP P450 nomenclature committee and tentatively named CYP345P1, CYP358B1, CYP4FD2, CYP4CD2 and CYP6JN1 under Gen Bank accession numbers: KT071005, KT071006, KT071007, KT071008 and KT071009, respectively. The length of the ORF ranged from 1,500-1554 bp. The length of the deduced amino acid sequences varied from 499 to 517 amino acids. Identity and protein BLAST analysis of the predicted amino acid sequences indicated the five genes belonged to three different families. We used the Mega 5.05 to generate the phylogenetic tree. The result showed that CYP4FD2, CYP4CD2 belong to CYP4 clade and CYP358B1, CYP345P1 and CYP6JN1 belong to the CYP3 clade.3 transcription profiles of P450 from L. entomophila3.1 Sexual expression profiles and developmental expression profilesthe m RNA levels of five CYP P450 genes and 12 Unigenes(Unigene 17618,Unigene 25300, Unigene 26646, Unigene 6837, Unigene 27020, Unigene 13178, Unigene 18998, Unigene 25537, Unigene 12385, Unigene 6029, Unigene 22015 and Unigene 25076) were expressed in both sexes and all developmental stages. The results showed that there were five P450 genes and 10 P450 Unigenes(Unigene 25300, Unigene 26646, Unigene 6837, Unigene 27020, Unigene 13178, Unigene 18998, Unigene 25537, Unigene 12385, Unigene 6029 and Unigene 22015) expressed more abundant in female than male adults. All P450 genes in our results were expressed at all tested developmental stages. There were differences in expression levels among different developmental stages. Expression of CYP345P1, CYP4CD2, CYP4CD2, CYP6JN1 and 9 Unigenes(Unigene 17618, Unigene 26646, Unigene 6837, Unigene 27020, Unigene 13178, Unigene 25537, Unigene 6029, Unigene 22015 and Unigene 25076)was higher in adults compared to other stages. It could infer that 13 P450 genes may play an important role in the adult stage. CYP358B1 and Unigene25300 expressed higher both in egg and adult stages than other stages, from which we infer that the two P450 genes may be involved in physiological activities and adaption to the environment.3.2 Insecticide induction expression profilesIn order to determine the response of P450 genes, three different insecticides,(deltamethrin, malathionand and propoxur) were chosen in this study. The q PCR results showed that P450 genes exhibited a rapid response under the exposure to malathion. Following exposure to malathion, the transcriptional patterns of the five genes were similar. The five CYP genes and 5 unigenes were up-regulated, reaching a peak of induction at 12 h, and the highest expression changes were for CYP4CD2 and CYP6JN1(9.77- and 9.47-fold increase respectively). This suggests that P450 genes expression changes rapidly after exposure to the organophosphate insecticides. As for deltamethrin, the five genes were up-regulated, reaching maximum expression at 36 h(the maximum of CYP358B1 was at 24 h). In addition,deltamethrin exposure resulted in the transcripts of Unigene 25076 and Unigene 22015 reached maximum expression at 24 h. the expression of Unigene 27020, Unigene 25537, Unigene 6029, Unigene 18998 and Unigene 6837 were down-regulated at 12 h. It could be indicated that these genes might participted in the detoxification process for deltamethrin of L. entomophila. Compared with malathion and deltamethrin, after propoxur exposure,the expression of CYP358B1 and CYP6JN1 were up-regulated. the transcripts of CYP345P1, CYP4FD2, CYP4CD2 and Unigene 25076 were down-regulated at first, then reached peak after 24 h. Unigene 17618、Unigene 25300、Unigene 26646、Unigene 13178、Unigene 18998 and Unigene 12385 increased up to the maximum value after 24 h.3.3 Expression Patterns among different geographical populationsThe relative expression was calculated based on the value of Suizhou expression level. The five P450 genes expressed more abundant in Tongliang than Suizhou(3.84-, 2.08-, 4.38-, 8.03- and 13.13-fold). The expression levels of CYP345P1 and CYP4CD2 were higher in Hannan population(5.00- and 6.37-fold than Suizhou).4 Comparision of P450 s activities in sensitive and resistant population from L. entomophilaWe determined the expression of P450 genes of sensitive and resistant population(Suizhou and Tongliang population) from L. entomophila. The results showed that the most P450 genes espressed aboudant in resistant population than sensitive population. And we assayed and compared the P450 s activities in sensitive and resistant population from L. entomophila. We found that the P450 activites of resistant populations were higher than susceptible population(1.93-fold). Under the insecticides stress, P450 activites in susceptible population raised compared with the control group. After induction of malathion and deltamethrin, the activities of P450 s increased 2.97- and 2.35-fold.5 Eukaryotic expression of P450 genes from L. entomophilaWe conducted the eukaryotic expression experiment with Bac-to-Bac system to explore the function of CYP4FD2. The recombinant virus expression vector Bacmid-CYP4FD2 was successfully constructed with the double digestion of Bam HI/ Not I restriction enzymes using the DNA recombination technology. Though, we did not finish transfecting Sf9 cells with the recombinant vectors, the generation of eukaryotic expression system for L. entomophila could make a contribution to investigate the specific function of P450 in our future experiment. |
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