The Functional Of Phospholipase C And E75 In 20E Pathway Of Apolygus Lucorum

Insect ecdysone is a steroid hormone secreted by the prothoracic gland of the insect.It can be activated into a biologically active 20-hydroxyecdysone?20E?catalyzed by P450monooxygenase in the hemolymph.Suede,and is in a critical starting position during life processes such as insect development,metamorphosis and reproduction.In the 20E signaling pathway,the classical nuclear receptor pathway has been identified,and 20E induces nuclear receptor ecdysone receptor?EcR?/ultraspiracle protein?USP?to form heterodimers and recruit Co-activators,thereby forming a transcriptional complex with DNA-binding activity,ultimately regulate transcription of genes downstream of 20E.Previous studies have shown that there is a membrane-mediated membrane receptor pathway in the 20E signaling pathway,which can control the 20E signaling pathway on the cell membrane.Phospholipase C?PLC?in the phosphoinositide metabolism system is involved in the regulation of the transduction of various hormones and neurotransmitters,but the biological characteristics of the genes and the response after 20E induction are not clear.In addition,E75 is an important downstream response gene of the 20E signaling pathway,and its genetic characteristics and its function in the regulation of 20E regulation of the Apolygus lucorum are still unclear.Therefore,in this paper,proteomic analysis,RACE,RNAi and other molecular biological methods were used to analyze the biochemical characteristics of membrane receptor PLC and nuclear receptor E75 in the 20E signaling pathway of the A.lucorum,and in the 20E signal transduction.The function was studied.Belows are the key research findings:1.In order to elucidate that the PLC can mediate the transmission of the 20E signaling pathway at the proteomics level,four different treatments?20E,20E+U73122,U73122 and acetone?of the Apolygus lucor?m were analyzed by iTRAQ method.The up or down of proteins abundance more than 2 folds as a standard threshold.Compared with the control acetone,the 20E treatment group had 100 differentially expressed proteins,of which 19 proteins were up-regulated and 81 proteins were down-regulated.The up-regulated proteins mainly have epidermal proteins,and down-regulated proteins mainly include ribosomal proteins and a small amount of energy metabolism-related enzymes.Compared with acetone treatment,U73122treatment group had 261 differential proteins,of which 42 proteins were up-regulated and 219proteins were down-regulated.Up-regulated proteins mainly contain different types of ribosomal proteins,and the down-regulated proteins mainly include epidermal proteins,allelochemical proteins,arginine kinases,salivary secretory proteins,and tissue proteins.Comparing 20E vs acetone with U73122 vs acetone,it was found that the two groups shared 20 different proteins.The expression of 20 proteins in each group showed the opposite trend,and all of them were ribosomal proteins.Analysis of the three treatments groups of 20E vs acetone,U73122 vs acetone,and 20E+U73122 vs acetone found that there were 20 differentially expressed proteins,but the proteins with opposite expression trends were mainly 5 ribosomal proteins.Then,the Venn diagram was drawn for the above three treatments,and it was found that there were 13 identical proteins in the 20E vs acetone group and U73122 vs acetone group,and the same protein contained 5 ribosomal proteins with opposite expression trends.Comparing 20E+U73122 with acetone,seven identical proteins were found.Comparing 20E+U73122 with acetone,seven identical proteins were found in Venn's diagram.Based on the results of differential protein expression and related studies reported,we speculate that 20E can induce the expression of PLC gene and then activate the downstream pathway IP₃-TOR-EcR/USP-Myc-ribosome biosynthesis after 20E treatment,which indicates that PLC participates in 20E signal transduction and further regulates the growth and development of the Apolygus lucor?m.2.The aims of this study are to obtain recombinant protein which has phospholipase enzyme activity and identify the best pH and the optimum temperature on the basis of cloned the phospholipase C from A.lucorum previous and analyzed the AlPLC_?with different expression of day age after the eggs hatch.In order to explore the basis for molecular regulatory mechanism.By the Real-time PCR technique,we determined the expression pattern of ALPLC_?in different day age of A.lucorum.To construct its prokaryotic expression vector.T-vector containing AlPlc was dual-enzyme digested by NdeI and XbaI,then the cDNA identified by sequencing was constructed to pCzn1 vector and transformed into Arctic express of E.coli.The target recombinant protein has over expressed and has been purified by using Ni-NTA agarose.Then its activity and enzymatic characteristics were studied by n-NPPC.qRT-PCR results showed that the A.lucorum ALPLC_?expression quantity in larvae 3?7?12 and 13 day of age is relatively high.The ALPLC_?gene could overexpress in E.coli and the target recombinant protein which purified through Ni-NTA agarose and had phosphodiesterase activity when using p-Nitrophenylphosphorylcholine as substrates and the most suitable reaction temperature was57??179.54�3.962 nmol/?g/min?,and the ideal pH was 8.7?374.99�2.838 nmol/?g/min?.This study analyzed ALPLC_?of A.lucorum in different day age mRNA expression quantity,constructed prokaryotic expression vector pCzn1-ALPLC_?,obtained with phospholipase activity of recombinant protein,and the suitable pH and temperature of the highest enzyme activity were8.5 and 57?,respectively.3.In order to further clarify the function of ALPLC_?in 20E signaling pathways,four treatments including 20E,20E+U73122,U73122 and acetone were designed to analyze the enzyme activity of ALPLC_?,and mRNA and protein expression,the content of the second messenger?IP₃ and DAG?is alsoanalyzed,and the six of the 20E signaling pathways downstream of the gene mRNA expression quantity change trend is studied.The results shows that 20E could induce the enzyme activity of AlPLC_?and its gene expression,while the enzyme activity of ALPLC_?and gene expression was significantly decreased under phospholipase C inhibitors U73122 treatment,in addition,the protein content of AlPLC?is in accordance with the above results by Western blot analysis.20E induced drastic accumulations of DAG and IP₃,which reached their peaks 1.5 h after the hormone treatment,while the accumulations of DAG and IP₃ were significantly decreased under U73122 treatment.In cultured midgut that were collected from newly emerged third nymph,expression of Tre-1?Tre-1?E75A?E75D?EcR-Aand EcR-B was induced readily by 20E.The 20E-induced expression of both genes was diminished remarkably when the PLC pathway were inactivated by PLC specific inhibitors U73122.4.A.lucorum is a predominant pest of Bacillus thuringiensis?Bt?cotton in China.The 20E plays a key role in the reproduction of insect.To better understand the mechanism underlying the20E-regulated reproduction,the nuclear hormone receptor E75 isoform-A of A.Lucorum?AlE75A?was cloned and expression analyzed.A 2241 bp sequence of AlE75A cDNA encoded an open reading frame of a polypeptide with a predicted molecular mass of 69.04 kD.AlE75A mRNA was detected in female adult stages of A.lucorum with a peak expression in the 7-day-old animals.AlE75A was also expressed in several tissues,especially maximal in the fat body and ovary.A 3.2 kb AlE75A mRNA was detected in all tissues by Northern blot.The fecundity and longevity were significantly decreased in female adults treated with AlE75A siRNA.The rates of egg incubation rates were considerably low in the RNAi-treated animals.In order to investigate the molecular mechanism underlying the effects described above,Vitellogenin?AlVg?was selected for further investigation.The expression pattern of AlVg was similar to that of AlE75A and was up-regulated by 20E.After knockdown of AlE75A,the expression profile of AlVg and the protein levels were downregulated.These findings suggest that AlE75A play a crucial role in the regulation of AlVg expression in A.lucorum.5.The E75 is one of the important early transcription factors in the molting process of insects.In this study,the cDNA of AlE75D was cloned from A.lucorum and its expression profile was investigated.We also obtained the recombinant proteins and prepared the highly specific monoclonal antibody against AlE75D protein.Using an RT/PCR approach followed by5?and 3?RACE,we isolated a 1911 bp sequence encoding a polypeptide with a predicted molecular mass of 75.68 kDa.Amino acid sequence comparisons indicated that the protein had the domain organization characteristic of a nuclear hormone receptor.That is,a highly conserved ligand-independent A/B activation domain?23 aa?;a hinge region?D domain,85 aa?;a ligand-binding domain?LBD??E domain,190 aa?which contained the putative ligand-dependent activation motif,and F domain?338 aa?.AlE75D was found to be continuously expressed through the whole adult stage of A.lucorum,and the expression of AlE75D reached its maximum value in the seven-day-old female adult.The transcript levels of AlE75D were higher in the fat body and ovary,in contrast,only small amounts of mRNAs for AlE75D were detected in flying muscle and Malpighian tubules.The vector containing AlE75D gene was double enzyme restricted by Nde I and XbaI,then the cDNA identified by sequencing was constructed to pCzn1vector and transformed to Arctic express of E.coli.The target recombinant protein has over expressed and has been purificated by using Ni-NTA agarose.BALB/c mice were immunized with the purified recombinant fusion protein.The spleen cells of mouse were fused with SP2/0cells.The specificity of hybridoma cell line was characterized by ELISA and Western-blot analysis,which could specificity combine with the total protein of A.lucorum and recombinant protein of AlE75D.