Biological Analysis Of Glutathione S-transferase And Small Heat Shock Protein Genes In Apis Cerana Cerana

Stress resistance is considered to be a crucial factor for honeybee population survival and continuity with the natural environment changes. The Asian honeybee (Apis cerana cerand) is an important indigenous species that plays an indispensable role in the balance of regional ecologies and agricultural economic development. Extensive studies have elucidated that GSTs are involved in regulating the antioxidant function in organism and sHSPs play key roles in the cellular defence against damages. In this study, we selected A. cerana cerana as the experiment material, and explored the biological functions about GSTO2and sHsp22.6genes and their relationships with the cellular defence. The main results and conclusions presented in this thesis are as follows:(1) Molecular characterization and functional analysis of GSTO2gene in A. cerana ceranaA novel Omega class GSTs gene in A. cerana cerana was cloned based on the conserved regions of GSTO from Apis mellifera Linnaeus by RT-PCR and RACE-PCR, and we named the putative gene AccGSTO2. The sequence analysis indicated that the full-length cDNA of AccGSTO2(JX434029) was1365base pair (bp), including a321bp5’untranslated region (UTR), a324bp3’UTR and a720bp complete ORE The ORF encoded a polypeptide of239amino acids with a predicted molecular mass (Mr) of27.785kDa and a theoretical isoelectric point (pi) of6.21. AccGSTO2shares higher sequence similarity with other insect species and contains the conserved features of the cytosolic GST superfamily.Using the TFSEARCH database, several transcription factor binding sites involved in tissue development and growth in early stages were identified in the5’-flanking region of AccGSTO2, such as CF2-II, BR-C and NIT2. Several important transcription factors required for regulating various environmental stresses (HSF and AP-1) and gene expression (CREB and C/EBP) were also found in AccGSTO2promoter region. These results suggest that AccGSTO2may be involved in organismal development and environmental stress responses.The qRT-PCR and Western blot were performed to examine the developmental regulation and tissue distribution of AccGSTO2. The highest expression level was detected in day-3larvae and in the brain, implying that AccGSTO2may play a protective role in the brain tissue and early development.The relative expressions of AccGSTO2under various biotic and abiotic stimulis were detected by qRTPCR. The results showed that the transcript levels of AccGSTO2can be markedly accumulated by treatment with various biotic stimulis such as Ascosphaera apis, Aspergillus flavus and20-hydroxyecdysone (20E) and abiotic stimulis such as temperature, UV, H2O2, pesticides and CdCl2. Whereas the transcription of this gene was gradually inhibited by HgCl2treatment. Western blot provided further evidence for the induced expression of AccGSTO2under CdCl2and microbe stressors at protein level, and the assay of oxidation statuses indicated that this induced expression of AccGSTO2may be directly related to the accumulations of H2O2and MDA. Taking into account these results, we speculate that AccGSTO2may be essential in protection against oxidative damage and tissue development.RNAi was employed to further study the biological functions of AccGSTO2gene. One-day post-emergence adults were injected with dsAccGSTO2or the third instar larvae were fed with additional dsAccGSTO2. The qRT-PCR displayed that lower transcript levels were observed in the dSAccGST02-injected adults at3day post-injection or in the dsAccGSTO2-fed larvae at1day post-fed. Immunohistochemical localization showed that the sections from dsAccGSTO2-tre&tment bees were more slightly stained than these from the controls, especially in the brain, muscle and midgut of adult and midgut, malpighian tubule and fat body of larvae. In addition, the knockdown of AccGSTO2caused a slight-increased AccP450mRNA level and marked reduction of transcriptional levels of AccGSTD, AccGSTs4, AccSOD and AccCAT. Moreover, compared to the control group, the AccGST02-RNAi bees had an increased H2O2and MDA accumulations, which revealed that the knockdown of AccGSTO2gene led to oxidative stress in vivo and thereby affected survival.The recombinant AccGSTO2protein was expressed using prokaryotic expression plasmid pET-30a(+) and the optimization expression condition. The soluble AccGSTO2protein was further purified by affinity chromatography to a final concentration1.89mg/mL. The purified enzyme showed GSH-conjugating activity towards CDNB and GSH peroxidaseactivity towards cumene hydroperoxide and t-butylhydroperoxide. Furthermore, the recombinant AccGSTO2showed a maximum DHAR activity at pH7.0and an optimum temperature of25℃.The mixed-function oxidation (MFO) system and disc diffusion assay were chosen to study the antioxidant function of AccGSTO2. In the MFO system, AccGSTO2protects gradually DNA from hydroxyl radical damage with its increasing concentrations, and the sulfhydryl moieties of cysteine residues in AccGSTO2may play important roles in DNA protection. Disc diffusion assay provides a further evidence for its protective effects in oxidative stress.Three AccGSTO2mutants were constructed by site-directed mutagenesis and named C28A, C124A, and C217A. The mutants C28A and C124A showed different loss of enzyme activity and shift of the emission maximum in kinetics and fluorescence spectra analyses. The MFO system and disc diffusion assay further demonstrated that Cys28and Cys124were responsible for implementing antioxidant activity. Homology modeling revealed that the mutant impacts may be caused by the changes of surface electropotential distributions, hydrophobic patches and spatial structure of proteins. We found that the properties of WT-AccGSTO2and the mutant C217A were roughly the same, indicating that Cys and Cys124may play important roles in maintaining enzyme structure and activity.(2) Molecular characterization and functional analysis of sHsp22.6gene in A. cerana ceranaA small Hsp gene in A. cerana cerana was isolated based on the above-mentioned method and was named AccsHsp22.6. The full-length904bp cDNA sequence of AccsHsp22.6(KF150016) included an115bp5’UTR, a204bp3’UTR and a585bp complete ORF. The predicted ORF encoded a194-amino acid protein with a computed theoretical pI of5.88and a Mr of22.605kDa. Multiple sequence alignments revealed higher sequence identity between AccsHSP22.6and other insect sHSPs. Moreover, the deduced AccsHSP22.6shares typical features of the sHSP family.In the5’-flanking region of AccsHsp22.6, except for some sequences involved in embryo or tissue development, including CF2-II, BR-C, NIT2, and CdxA, several important transcription factors associated with environmental stress and immune response, such as HSF, AP-1, Nrf2, NFκB and p53, were also found using the MatInspector database. These putative transcription factor binding sites imply that AccsHsp22.6may be involved in not only environmental stress responses but also developmental regulation.The qRT-PCR and Western blot were performed to determine temporal and spatial expression profiles of AccsHsp2’2.6. We noticed that a significant increase of AccsHsp22.6expression always correlated with the transition of different development stages. The highest expression level was detected in the newly emerged adults and in the midgut. These results indicate that AccsHsp22.6might be involved in the control of honeybee development and protecting against the damage of xenobiotics.The expressions profiles of AccsHsp22.6under various experiment conditions were detected by qRTPCR. We found that the transcript levels of AccsHsp22.6can be markedly up-regulated by treatment with various abiotic stresses such as heat, cold, UV, H2O2, cyhalothrin and CdCl2and biotic stresses such as Ascosphaera apis, Aspergillus flavus and20E. However, the transcription of this gene showed fluctuating and suppressed pattern during25℃, phoxim, paraquat, and HgCl2stress. These observations provide clues for the possible protective role of AccsHsp22.6in the physiological dysfunctions evoked by the major adverse factors.The biological functions of AccsHsp22.6gene was further studied by RNAi. After one-day post-emergence adults were injected with dsAccsHsp22.6, qRT-PCR displayed that significant transcriptional suppression could be observed in the dsAccsHsp22.6-inected group, especially at1-3days after injection. The sections from dsAccsHsp22.6-treated honeybees were more slightly stained than those of the controls, especially in the midgut and fat body especially in the midgut, malpighian tubules, muscle and fat body of adult, although without visible morphologic alteration. Additionally, we found that the AccsHsp22.6-RNAi honeybees after RNAi treatment for3days had lower survival rate than that of the control honeybees during4℃or50℃treatment. These finding suggest that the inhibition mediated by dsAccsHsp22.6is effective, and the knockdown of AccsHsp22.6may weaken cellular thermotolerance in extreme temperature stress.The recombinant AccsHSP22.6protein was expressed by prokaryotic expression plasmid pET-30a(+) and the optimization expression condition. The soluble recombinant protein was further purified using the affinity chromatography columns, the concentration of the purified AccsHSP22.6was approximately1.67mg/mL. The molecular chaperone activity analysis showed that the purified AccsHSP22.6effectively protected malate dehydrogenase (MDH) from heat-induced denaturation in vitro. Escherzchia coli overexpressing AccsHsp22.6displayed a higher survival rate to4℃and50℃stresses than cells containing only the pET-30a(+) vector, indicated that AccsHsp22.6expression in E. coli cells enhanced cellular thermotolerance. A disc diffusion assay also provided evidence in vivo for AccsHSP22.6’s protective roles in oxidative stress.