| Bemisia tabaci(Gennadius)is an important pest worldwide.Beauveria bassiana as an important entomopathogenic fungus,could infect and kill the insect.Meanwhile,immune response ininfected B.tabaci could impede the infection process.Immunity of B.tabaci could affect infection efficiency of fungi,which always is the hot topic in the fields of pest management with entomopathogenic fungi.In this paper,tgenes of B.tabaci ?-1,3-glucan recognition proteins(Bt?GRPs),serine protease(BtCLIPs)and lysozyme(BtLyzs),which involved in immune response,were identified and analysed.The amino acid sequences translated form the genes were phylogenetic analysed with homologous proteins in other insects.the expression of genes with time in fungi-infected B.tabaci were analyzed by quantitative real-time PCR.Furthermore,the prokaryotic expression and purification of Bt?GRPs were obtained which could be used in further study.This study laid the foundation of exploring novel target for biocontrol of B.tabaci,and enhancing the role of B.bassiana in the IPM system of B.tabaci.The major results and conclusions are listed as follows: 1.Innate immune response of the pattern recognition receptor ?GRPs in B.tabaciIn order to understand the roles of ?-1,3-glucan recognition protein(?GRP)in the signal pathways of innate immune reaction in B.tabaci,three ?GRPs identified in B.tabaci were characterized by using protein structure analysis and phylogenetic analysis with homologous genes of 23 other insects.The different developmental stages of B.tabaci were infected by the fungus B.bassiana and subsequently the expression of ?GRP genes was analyzed by quantitative real-time PCR.The protein structure analysis showed that three sequences had the unique structure of ?GRP family which contained conserved domains-glycoside hydrolase activity domain.Three Bt?GRPs formed a clade specific to B.tabaci that would be involved in a unique cascade path of B.tabaci.Transcriptional levels of three ?GRPs genes in B.tabaci were up-regulated at different stages and different time points compared to the control,except Bt?GRP3(not expressed in the egg stage),but the degree of infection was different.The relative expression quantity peaked in adults at 24-36 h,later than in eggs and nymphs,probably due to wax and other substances on the surface of B.tabaci,which delayed the infection of B.bassiana.In conclusion,the three Bt?GRPs might participate in the innate immune responses to the infection of the fungus in B.tabaci,and could be a novel target for biocontrol of B.tabaci.2.Analysis on clip serine protease genes involved in innate immunity of B.tabaciTo study clip-domain serine proteases in tobacco whitefly B.tabaci,eight CLIP serine proteases identified in B.tabaci were analyzed by protein sequence alignment and phylogenetic analysis with homologous CLIPs in Anopheles gambiae,Bombyx mori,Drosophila melanogaster and Manduca sexta.The 4th instar B.tabaci nymphs were infected by Beauveria bassiana and the expression of clips serine protease genes was subsequently analyzed by qRT-PCR.The results showed that the eight BtCLIPs genes contained CLIP domain;BtCLIP19,BtCLIP20 and BtCLIP21 formed a specific clade.The other BtCLIPs were evolutionarily close to one or more homologous serine proteases in the four model insects.The transcriptions of eight BtCLIPs genes in B.tabaci infected by B.bassiana were up-regulated and peaked at 48 h.The transcription of BtCLIP19 was the highest among all BtCLIPs genes and increased 71 folds compared to the control.3.Identification and expression analysis on lysozyme gene of B.tabaciFour lysozyme genes were identified and designated as BtLyz1,BtLyz2,BtLyz3 and BtLyz4,which were 1 819,1 149,829,928 nt,respectively.They were predicted to encode proteins of 159,160,148,160 amino acids,respectively.Amino acid sequence alignment,phylogenetic analysis and protein tertiary structure prediction showed that Bt Lyz1 and BtLyz4 belong to i-type lysozymes,BtLyz2 and BtLyz3 belong to c-type lysozymes.BtLyz-1 formed a clade specific with Acyrthosiphon pisum of ApLyz-i1,BtLyz4 formed a clade specific with Diaphorina citri of DcLyz-i3 and Acyrthosiphon pisum of ApLyz-i2;BtLyz2 and BtLyz3 formed a clade specific with Nilaparvata lugens of NlLyz-c1 and Harmonia axyridis of HaLyz-c3.Compared to the control,the relative expression of all four genes in egg and BtLyz4 in nymph were not induced significantly by fungi-infected;the transcription of BtLyz1 nymph stage underwent the fluctuation of up-regulation,down-regulation,and second up-regulation and peaked at 24 h,it was increased 4.55 folds compared to the control;the transcription of BtLyz1 and BtLyz4 adult stage underwent the fluctuation of up-regulation,down-regulation,and second up-regulation and peaked at 60 h,they were increased 11.31 and 4.21 folds compared to the control.The transcription of BtLyz2 and BtLyz3 nymph stage underwent the fluctuation of up-regulation and down-regulation and peaked at 24 h,they were increased 5.09 and 8.31 folds compared to the control,then down-regulated obviously at 60 h,they were reduced 0.19 and 0.13 folds compared to the control.The transcription of BtLyz2 and BtLyz3 adult stage underwent the fluctuation of up-regulation and down-regulation and peaked at 24 h,they were increased 5.56 and 8.84 folds compared to the control.Four BtLyz genes were identified in B.tabaci.Among them,two are c-type and two are i-type lysozymes.Transcriptional levels of four BtLyzs genes in B.tabaci were induced through different developmental stages and different time points in fungi-treated individuals compared to the control,they were not induced significantly in eggs,and showed different expression trends in nymphs and adults.4.Expression and purification of the pattern recognition receptor ?GRPs in B.tabaciIn this study,we used the results of the second-generation high-throughput sequencing to amplify Bt?GRP1 and Bt?GRP2 ORF.The Bt?GRP1 contains a 837 bp open-reading frame encoding a putative protein of 278 amino acids and the Bt?GRP2 contains a 531 bp open-reading frame encoding a putative protein of 176 amino acids.We also constructed the recombinant expression plasmid pET-30a(+)/Bt?GRP1 and pET-32b(+)/Bt?GRP2 and the recombinant gene was transferred into competent cells,IPTG was then added to the culture and found that pET-30a(+)/ Bt?GRP1 and pET-32b(+)/ Bt?GRP2 were expressed in large quantities and almost unchanged,the two kinds of protein were expressed in sediment and short expressed in supernatant.The concentration of 0.2mmol / L to induce expression of the target products for approximately 6 h was selected.The target protein was present in the form of precipitation using SDS-PAGE and the aim proteins were purified using a Ni-NTA-Sepharose Column,the molecular weight of about 40 kDa and 28 kDa,respectively was consistent with the fragments in the process of digestion.The recombinant proteins were dtermined with the method of BCA protein concentration determination and the concentrations of Bt?GRP1and Bt?GRP2 were 1.893 mg/mL and 2.138 mg/mL,respectively which was satisfied with the following experiment.The target protein were sent to the company for recombinant protein antibody preparation,which was used for the following protein function detection. |
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