Regulatory Mechanism Of CYP6B6 Responding To 2-tridecanone In Helicoverpa Armigera

Insect cytochrome P450 enzymes（CYP）play an important role in the decomposition and metabolism processes of toxic substances.The existing researches show that P450 can be induced by pesticides,medicines and chemical substances.The expression and synthesis of P450 can be induced by plant secondary metabolites when cotton bollworm feeding on plants.Those enzymes metabolize exogenous secondary substances and pesticides.In the cotton bollworm CYP450 superfamily,the expression of CYP6B genes plays an important role in the regulation of plant secondary metabolites.And CYP6B genes’overexpression causes bollworm resistance to insecticides.The experimental study found that the plant secondary metabolites 2-tridecanone（2-TD）induced the cotton bollworm（Helicoverpa armigera）larvae CYP6B6 gene overexpression,reduced the larval weight and 20E titer,prolonged the pupated time and reduced the pupation and eclosion rate of cotton bollworm after 10 days of 2-TD treatment.However,artificial diets containing CYP6B6 ds RNA could reduce the expression of CYP6B6,reduce the larval weight,shorten the larval length,prolong the larval stage,hinder larval moult and even cause larval death.These results indicate that CYP6B6 may be involved in the metabolism of ecdysone and juvenile hormones in the cotton bollworm.In this paper,the 6^th instar larvae of H.armigera were treated with 10 mg/g 2-TD for different time,combined with Yeast one-Hybrid（Y1H）technology and Transcriptome Sequencing（RNA-seq）technology to obtain candidate regulatory factors that induce CYP6B6 expression,and preliminary understand the metabolic pathways about growth and development of cotton bollworm after 2-TD stressing.The fusion proteins of candidate regulatory factors were expressed using the Escherichia coli system,and the temporal and spatial expression profile of candidate regulatory factors was detected in the larval stage of H.armigera by Real-time Quantitative RT-PCR（q PCR）.The regulatory factors bond to the 2-TD responsive element in CYP6B6promoter,which was HE1 element,was determined by Electrophoretic Mobility Shift Assay（EMSA）technique.The expression of each regulatory factor after 2-TD or CYP6B6 ds RNA treatment was determined by q PCR and correlation analysis.And last,Yeast two-Hybrid（Y2H）technique was used to identify the protein-protein interactions between regulatory factors.The all results of them preliminarily mastered the pathway of CYP6B6 expression induced by regulatory factors after 2-TD treatment,which provided a theoretical basis for CYP6B6 regulation of the growth and development of H.armigera,and screened the key genes about the growth and development of H.armigera.And last,it can establish an efficient,stable and environment-friendly pest control methods.1.Y1H screens candidate regulatory factors for 2-TD induced CYP6B6 expressionUsing 10 mg/g 2-TD treated the 6^th instar larvae of H.armigera,and mixed c DNA libraries were established at different times（4 h,9 h,17 h,22 h,29 h,33 h,and 41 h）.A bait reporter strain was constructed in response to the 2-TD core sequence of CYP6B6promoter.Y1H system was used to screen the interacting protein factors with c DNA library and the bait reporter strain.After multiple times of Ab A screening,29 positive clones related to 2-TD induction were obtained.The plasmids of these yeasts were transformed into E.coli DH5αand sequenced to find that they belonged to 17 different protein sequences.The transcriptional activation of these 17 sequences in the reporter strain was verified.Finally,six proteins involved in the 2-TD stressed CYP6B6expression process were identified,that is,CYP6B6 responded to 2-TD induced candidate regulatory factors.They were phosphatidylethanolamine-binding protein（PEBP）,lipopolysaccharide induced tumor necrosis factor alpha factor（LITAF）,eukaryotic translation initiation factor 3 subunit G（e IF3G）,alcohol dehydrogenase 5（ADH5c）,calponin（CAL）and odorant receptor protein（OR）.2.RNA-seq clears the metabolic pathways after 2-TD inductionAfter treatment with 10 mg/g 2-TD for 6 h and 15 h,c DNA libraries were obtained from midgut of 6^th instar larvae of H.armigera.Using RNA-seq analyzed the expression patterns of all genes in the c DNA libraries.From this,2-TD induced differentially expressed genes（DEGs）were obtained,and the molecular functions,biological processes,and cell composition of DEGs were analyzed to grasp the2-TD-induced metabolic and signaling pathways.There were 380 DEGs（210up-regulated and 170 down-regulated）after treated 6 h and 314 DEGs（177 up-regulated and 137 down-regulated）after treated 15 h.These DEGs were significantly enriched in biological,metabolic,and cellular modified amino acid metabolic processes,which mainly molecular functions were catalytic activity,oxidoreductase activity,iron ion binding,heme binding,tetrapyrrole binding,acyl-Co A dehydrogenase activity and transferring hexosyl group transferase activity.Thereby participate in the Fatty acid degradation,Metabolism of xenobiotics by cytochrome P450,Insect hormones synthesis,Valine/leucine/isoleucine degradation Glutathione metabolism,Peroxisome and Riboflavin metabolism,and Jak-STAT signaling,m TOR signaling,Fox O signaling,Phototransduction-fly and other pathways.The DEGs such as LITAF,e IF,ADH,FK506-binding protein（FKBP）and forkhead protein（FOXA）were found to participate in 2-TD induced growth and development of H.armigera.3.Cloning,prokaryotic expression of candidate regulators and the temporal and spatial expression in H.armigeraThe candidate regulatory factors were amplified using the midgut c DNA of 6^thinstar larvae of cotton bollworm with 10mg/g 2-TD treated.The nucleic acid sequences of them were obtained and transformed into E.coli system to express the fusion protein.And q PCR was used to detect the expression levels of various candidate factors in different stages of larvae and different tissues of 6^th instar larvae of H.armigera.The results showed that the open reading frame（ORF）sequences of Ha PEBP,Ha LITAFa,Ha LITAFb,Hae IF3G,Ha ADH5b,Ha ADH5c,Ha CAL,Ha FABP1,Ha FKBP12,and Ha FOXA2 were 588 bp,342 bp,237 bp,837 bp,1005 bp,1005 bp,564 bp,405 bp,327bp and 669 bp,respectively,encoding 195,113,78,278,334,334,187,134,108 and222 amino acids.Spatial and temporal expression of 10 candidate regulatory factors showed that all candidate regulators except Ha FOXA2 were expressed in all stages of larvae and were high in older larvae and prepupae,while the remaining 8 candidate regulators except Ha LITAFa and Ha FOXA2 were all expressed in the fat body,midgut,head and integument of 6^th instar larvae,and the high expression were in the fat body and midgut4.EMSA,2-TD treated and CYP6B6-RNAi to obtain regulators that bind to 2-TD core sequence of CYP6B6 promoterThe EMSA was used to verify whether the candidate regulators Ha PEBP,Ha LITAFa,Ha LITAFb,Hae IF3G,Ha ADH5b,Ha ADH5c,Ha CAL,Ha FABP1,Ha FKBP12,and Ha FOXA2 interacted with the 2-TD core sequence of CYP6B6promoter that is HE1 element to identify the true regulators.The expression of regulatory factors was detected by 2-TD induction and CYP6B6 silencing to assume the combination mode with the HE1 element.The results showed that 7 regulators Ha LITAFa,Ha LITAFb,Hae IF3G,Ha ADH5c,Ha CAL,Ha FKBP12 and Ha FOXA2could bind to the HE1 element and they were the regulators to induce CYP6B6expression.The genuine regulators were induced after 10 mg/g 2-TD treated for different time.Although the expression level of them was not the same,the expression patterns of CYP6B6,Ha LITAF and Ha FKBP were similar that increased at 12 h to the maximum and then decreased to reach the minimum at 48 h,and the expression patterns of Ha ADH5c and Ha CAL were consistent that reduced from the 6 h maximum to the 48h minimum,while that of Hae IF3G和Ha FOXA2 were alike which was lower in the 20h and higher at 48 h than the control group after 2-TD treated.The correlation analysis was performed on the expression of each regulator after induction with 10 mg/g 2-TD.There were three highly positive correlations,which were the one CYP6B6 and Ha LITAF,another Ha ADH5c and Ha CAL and the other Hae IF3G and Ha FOXA2.And only Ha FKBP and Ha FOXA2 was highly negative correlation.The expression of all regulators except Ha FOXA2 was significantly decreased at 48 h and 72 h in the CYP6B6 silencing treatment,while the transcripts were slightly tested not in control group but in the CYP6B6 silencing group.All above results showed that the regulators,which were Ha LITAF,Hae IF3G,Ha ADH5c,Ha CAL,Ha FKBP12 and Ha FOXA2,together controlled the expression of CYP6B6,thereby participating in the cell growth and development pathway in the bollworm.5.Y2H tested the interaction between regulatory factorsThe ORFs of the regulators（Ha LITAF,Hae IF3G,Ha ADH5c,Ha CAL,Ha FKBP and Ha FOXA2）were linked to the two yeast expression vectors p GBKT7 and p GADT7.The recombinant plasmid p GBKT7-prey was transformed into the yeast Y2HGold strain.Afterwards,the self-activation effect of each factor was detected by the deficient medium SDO/X.Then the two recombinant plasmids were co-transformed into the yeast Y2HGold strain,and the two-hybrid recombinant strain Y2HGold（p GBKT7-prey）（p GADT7-prey）was obtained.The growth and coloration status of the two-hybrid recombinant strain was verified by defective media DDO/X and QDO/X/A.The results showed that all the Y2HGold（p GBKT7-prey）strains normally grew on SDO/X,and only Y2HGold（p GBKT7-Ha FOXA2）strain was blue.It meant that except Ha FOXA2 other factors’ORF could not lonely activate yeast transcription.Only Y2HGold（p GBDT7-Ha PEBP）（p GADT7-Hae IF3G）,Y2HGold（p GBKT7-Ha LITAF）（p GADT7-Hae IF3G）and Y2HGold（p GBKT7-Hae IF3G）（p GADT7-Hae IF3G）strains were able to grow on DDO/X and QDO/X/A.Both normal growth and displayed blue indicated that the protein-pretein interaction as Ha PEBP-Hae IF3G,Ha LITAFb-Hae IF3G and Hae IF3G-Hae IF3G activated yeast transcription,while Ha PEBP might be a cofactor to participate in the process of CYP6B6 expression under2-TD stressed.