Mechanism Of SeGrx1 In Spodoptera Exigua Regulating Oxidative Stress And Apoptosis Induced By CPT

In our previous study,we found that Spodoptera exigua midgut cell lines IOZCAS-SPEX-?exposure to camptothecin?CPT?would result in cell apoptosis characterized by the accumulation of a large intracellular ROS,the damage of protein,lipid and DNA,calcium influx,the decrease of mitochondrial membrane potential.Subsequently,the result of transcriptional sequencing showed that the expression of glutenin 1?Grx1?in Spodoptera exigua changed in apoptosis process.Therefore,it is speculated that Grx1 of S.exigua may be involved in oxidative stress damage caused by CPT treatment and modification and signal transduction of many proteins in the process of apoptosis,which regulated the process of oxidative stress and apoptosis.However,the basic biological characteristics and antioxidant mechanism of Grx1 have not been systematically and deeply studied.In order to uncover the regulatory function and mechanism of Grx1 in the process of insect oxidative stress and apoptosis,and the combining using of dsRNA of Grx1 gene and CPT to explore a new ecologically and safe pest control strategies.In this study,camptothecin was used as the inducer of oxidative stress and apoptosis of S.exigua midgut cell line IOZCAS-SPEX-?.With rapid-amplification of cDNA ends cloning technology,prokaryotic expression and purification of protein,RNA interference,transcriptome sequencing,qPCR and other experimental methods,we analyzed the structure of Grx1 gene of S.exigua,explored the interacting protein of SeGrx1,determined its redox catalytic activity and the function and mechanisms of Grx1 in the regulation of apoptosis induced by CPT.The main results are as follows:The essential biological characteristics analysis of SeGrx1 gene.The full length of SeGrx1 gene was cloned by RACE technology.The full length of SeGrx-1 gene in S.exigua is 738 bp?GenBank accession number:MK318813?.The open reading frame?ORF?is 351 bp that encoded 116 amino acids.The relative molecular weight of the predicted protein is 12.57 kD.Through bioinformatics analysis,it was found that SeGrx1 protein contains a classical KXXCPYC catalytic site,which belongs to double sulfhydryl glutaredoxin.At the same time,the conservation of catalytic site and GSH binding pocket amino acids also shows that SeGrx1 has a typical glutamate redox protease activity.SeGrx1 has redox activity against substrate glutathione reductase and plays an important role in protecting insect tissues from oxidative stress.The kinetic parameters of enzymatic reactions for purified S.exigua SeGrx1.The prokaryotic protein expression vector of SeGrx1 was constructed.SeGrx1 was successfully expressed in vitro through continuous optimization of induction and purification conditions,and the purity of SeGrx1 target protein was more than 95%.The specific activity of SeGrx1 was 0.577 U/mg pro,the maximum reaction rate V_max was 20.5 U/mg pro,and the Michaelis constants Km was 14.3 nM.It was found that the enzyme activity and catalytic activity are relatively high,which paved a theoretical basis information for further exploring the biological function of glutamate and mastering its role in insect life activities such as oxidative stress and apoptosis.The function and mechanisms of SeGrx1 in the regulation of apoptosis induced in S.exigua by CPT.RNAi technology was used to interfere with the expression of SeGrx1 gene.It was found that SeGrx1gene was highly sensitive to oxidative stress of CPT.Cells protected cells from oxidative stress and a_ap_nto_ip_ot_xo_is_di_as _n i_t n_sd_yu_stc_ee_md._Mi^byy _tC_ocP_hT_{o n}b_dy_{r i a}m_l ai_mn_et_ra_ci_ani_pn_tag _{n r}^a _e p_gr_uo_lp_ate_or _{r y}l _devi^evelvel _co_ef _wR_aO_s S_f,_{o r}t_mhu_es_{d w}m_ia_ti_hn _thioal^taining _c t_oh_he_{o l} b_mal_oa_ln_ec_ce_{u l} o_ef_s such as Grx,Trx and Prx,which regulated the function of important mitochondrial enzymes to respond to the changes of mitochondrial redox state.Exploring the interacting protein with SeGrx1 in Spodoptera exigua.The transcriptome of midgut cells of beet armyworm was sequenced and bioinformation analysis,the cells with interfered expression of SeGrx1 gene were exposure to 10?M CPT.The results showed that 6353 genes were up-regulated and7296 genes were down-regulated.Comparing with normal cells treated with 10?M CPT,there were 5210unique differential genes.The 26 out of 5210 differential genes were selected for real-time fluorescence quantitative PCR verification.It was found that there were significant differences in CPY4c1,Uqcrc2,Txn2,Prdx5,Timm22,AGAP0199,chchd4 and GstD1,which were all mitochondrial redox related genes.These mitochondrial redox related proteins offered basic information for illustrating the pathway of regulation in oxidative stress and aopotosis in insects.At the same time,BNIP3 and ASK1 were found in the specific differential gene pool.The full length of SeBNIP3 gene?GenBank accession number:MK318815?was coloned,the ORF was 582bp that encoded 193 amino acids,SeBNIP3 contains only one BH3 domain,which was predicted to promote apoptosis;The full length of apoptosis-induced kinase SeASK1 gene?GenBank accession number:MK318816?was cloned,and the ORF was 4176 bp that encoded 1391 amino acids;At the same time,the full length of the same family gene SeGrx3?GenBank accession number:MK318814?was cloned,and the ORF was 663 bp that encoded 220 amino acids,it was predicted that SeGrx3 of S.exigua may be involved in iron metabolism and oxidative stress regulation.Combining with transcriptome data and functional prediction analysis,SeGrx3,SeBNIP3 and SeASK1are involved in the regulation of oxidative stress and apoptosis in insects,and may interact with SeGrx1.The influence of SeGrx1 on insecticidal activity of CPT against Spodoptera exigua.The inhibitory effects of dsRNA of SeGrx1 combined with plant secondary metabolites CPT,compared with CPT and dsRNA alone on S.exigua.The results showed that the combining using of dsRNA and CPT had more significant inhibitory effect on beet armyworm.The attempt of this study provided an innovative strategy for the combined using of RNAi and CPT in the field of lepidoptera insect control.Taken with the above studies,SeGrx-1 protein was successfully expressed in vitro,and its enzyme-kinetic parameters were obtained,which paved a basis information for further exploring the biological function of glutaredoxin and its application in pest control.Through the construction of mitochondrial mercaptan signal regulatory device,SeGrx1 plays an important role in maintaining the balance of antioxidant system,protecting cells from oxidative stress and resisting apoptosis induced by CPT,which provides an innovative strategy for RNAi technology combined with CPT to control lepidoptera insects.