Research On Mechanisms Of Bactrocera Dorsalis(Hendel) In Response To Thermal Stress

The oriental fruit fly, Bactrocera dorsalis (Diptera, Tephritidae) is an important pest. It distributes world widely and causes severe economic losses on more than250species of fruits and vegetables among46families. B. dorsalis presents the strong abilities to environmental adaptation and geographic expansion, which can invade from long scale distance and quickly take over the hosts. This insect had been firstly found in tropical and subtropical areas, and now it has become a dangerous fruits and vegetables'pest in south China, Southeast Asia, India subcontinent and Hawaiian Islands, where the temperature is sometimes higher than40℃in summer. As the global warming, B. dorsalis has gradually expanded to high latitudes, and could overwinter in the areas of Hubei, Jiangsu, and other provinces in China, with the temperature lower than0℃in winter. Normally, the appropriate development temperature range of B. dorsalis is from15to34℃, so the extreme temperatures of summer and winter would cause thermal stress to oriental fruit fly. It results that B. dorsalis has formed the series of response mechanisms to cope with heat stress from living environments. Heat shock proteins (Hsps) are the most conservative proteins in the biological evolution, encoded by the heat shock genes and generated by cells or organisms under thermal or other environmental stresses. As accompanied proteins, Hsps are significantly related to organism resistance to environmental stresses. Oxidative damage caused by thermal stress would lead to peroxidation reaction in vivo, and antioxidant systems can scavenge oxygen free radicals, alleviate oxidative stress and repair stress injury. The development of proteomics, supplies the favorable conditions to isolate and identify the proteins of insects, with core technologies of two-dimensional electrophoresis and mass spectrometry. The rise of comparative proteomic provides a powerful tool for the separation of these known thermal stress proteins, as well as to explore the role of unknown proteins involved in thermal stress response process.This dissertation aimed at the international research foci on B. dorsalis biological adaptation to thermal stress and carried out the research on comparative proteomics under thermal stress to separate response proteins and identified them. The research also measured the activities changes of antioxidant enzymes under thermal stress. The molecular cloning and characterization, and transcriptional expression profiles of relative Hsps and antioxidant enzyme genes under thermal stress were carried out too. The study provides the comprehensive analysis of the response mechanisms of B. dorsalis to thermal stress. The main results obtained were as follows:1. Antioxidant response of B. dorsalis to thermal stressMalondialdehyde (MDA) is the main oxidation product in the multi-unsaturated fatty acid peroxidation, which has been used as a biomarker of oxidative stress through the measurement of its content to determine lipid peroxidation (LPO) level. The MDA content was significantly increased under the high or low extreme temperature stress of the B. dorsalis compared with the control27℃, which proved that the thermal stress broke the balance of oxidation-reduction reaction of B. dorsalis, and induced the increase of antioxidant enzyme activities. The activities of superoxide dismutase (SOD) significantly increased under the thermal stress, which catalyzed the disproportionation reaction of high concentration of superoxide anion (O2·-) generated under oxidative stress, producing hydrogen peroxide (H2O2). The activities of SOD rose as the trend of the temperature approached the extreme. With the duration of thermal stress, the activities of SOD increased at first, and then decreased. The activities of catalase (CAT) was significantly higher than control, which could help to decompose excess H2O2into harmless H2O and O2in order to ease the oxidation stress by reducing the reactive oxygen species (ROS). As the trend of the temperature closing to the extreme, the activities of CAT increased, and the activities of CAT increased at first, then declined as the duration of stress. The activity of glutathione S-transferase (GST) has also undergone significant changes under thermal stress, which significantly increased under high temperature stress, and decreased after the rapid increase compared with control27℃during the extension of the stress time under low temperature stress. The increased GST activities could decompose the excess lipid peroxides, and play an important role in B. dorsalis oxidative stress reactions during thermal stress. The higher value of the activities of Peroxidase (POD) and total antioxidant capacity (T-AOC) play stably roles in resistance to oxidative stress.2. Comparative proteomic researches of B. dorsalis under thermal stressTwo-dimensional electrophoresis is an efficient separation technology to the complex organism protein system, and the resolution, reproducibility and stability are the three core requirements. The protein sample preparation is a critical first step which poses a direct impact on the quality of the protein samples separation and the integrity of biological information. Total proteins from the whole body of B. dorsalis were used in order to get fully insight of response proteins under thermal stress. In this study,2M thiourea was added in the lysis buffer to increase the solubility of proteins, especially to hydrophobic proteins. In order to obtain the purified protein samples, the purification kit from Bio-Rad was used to remove interfering substances, such as phenols pollution. So, the crucial first step for the subsequent two-dimensional electrophoresis has been performed. The concentration of glycerol and SDS were enhanced in balance buffer, boosted the strip balance effect. Silver staining method was adapted to PAGE gel staining, which resulted in high-resolution two-dimensional gels. The establishment of the approach to a stable and repeatable two-dimensional electrophoresis system provided the foundation for the separation and identification of proteins to B. dorsalis thermal stress response.Using proteomics technology to study the B. dorsalis thermal stress response mechanisms, the total proteins were extracted from treatments of thermal stress under40and0℃for3h,27℃as control. The two-dimensional electrophoresis experiments were conducted using strips of pH3～10, length17cm and10%SDS-PAGE gel. Three groups of two-dimensional gel electrophoresis have the high match rate according to PDQuest V8.0.1software analysis. Compared with the control27℃, we used the automatic matching function of PDQuest and combined with artificial increase and removal of the protein spots,61diversity protein spots were identified successfully. Particularly,37spots were consistently expressed under thermal stress, while30of them up-regulated and7of them down regulated. According to MALDI-TOF-TOF tandem mass spectrometry sequencing analysis,40proteins have been successfully identified. These proteins are15oxidoreductases, such as Superoxide dismutase, Peroxiredoxin1, Cytochrome P450and Respiratory-chain NADH dehydrogenase,8various types transferase of ions and metabolites, such as Peptide Revolution-O-fucosyltransferase, Glutathione S-transferase, Guanido phosphotransferase, and so on. There are also a variety of kinases and mitochondrial glycoprotein as well as Hsps. The identification of these proteins provides a powerful insight to understand the thermal stress response of B. dorsalis from the protein level.3. Molecular cloning, sequence analysis and mRNA expression profiles of B. dorsalis Hsps genesBy using RT-PCR and RACE, full-length of four Hsps gene were cloned, including one gene from Hsp90family (named as:BdHsp901), two genes from Hsp70family (named as BD1and BD2), and one gene from Hsp60family (named as Hsp60). All these four Hsps genes were submitted to GenBank. As many as other insects'Hsps amino acid sequences were downloaded from GenBank to generate the phylogenetic tree to determine these four Hsps genes'classification using the software MEGA4.0(the Neighbor-joining method). The expression profiles of these four Hsps under thermal stress were determined by real-time fluorescence quantitative qPCR technology, with actin gene (GenBank accession number L12254) as the internal reference.The full length cDNA of BdHsp901(GenBank accession number JN168997) was2621bp, with the open reading frame of2148bp, and encoding715amino acids. It had an Hsp90family signature sequence YSNKEIFLRE, and it was determined as the cytoplasmic Hsp according to the C-terminal motif MEEVD. Based on the obtained phylogenetic tree, the amino acid sequences encoded by BdHsp901showed the closest relationship with Mediterranean fruit fly Ceratitis capitata, which were clustered into one, and then gathered with the Tsetse fly Glossina morsitans, while presented the far distant with the Lepidoptera. The expression of BdHsp901was significantly increased after high temperature stress for3h, which suggested that B. dorsalis could accumulate proteins encoded by this gene to adapt to pressures from the high temperature of environment. Normally, Hsp90family are one of highly expressed proteins in all organisms, which was only significantly up-regulated in the low temperature stress of-5℃for3h, indicating that the amount of BdHsp901could protect cells from stress damage in gentler low-temperature (>-5℃).The GenBank accession numbers of two Hsp70genes are GU591408and GU591409, respectively. The full-length cDNA of BD1(GenBank accession number GU591408) was2086bp, with an open reading frame of1962bp, and encoding653amino acids. It had three of the Hsp70family signature sequence IDLGTTYS, IFDLGGGTFDVSIL and IVLVGGSTRIPKVQR. The full-length cDNA of BD2was2258bp, with the open reading frame of1911bp, and encoding636amino acids. There were only two Hsp70family signature sequence, IDLGTTYS and IFDLGGGTFDVSIL, in the amino acid of BD2. Both genes showed non-organelle motif RARFEEL, highly conserved C-terminal cytoplasmic Hsps motif EEVD, as well as two-component nuclear localization signal, KK and RRLRT. The results of the phylogenetic tree analysis showed that the two Hsp70family genes were clustered with Tephritidae (Diptera). The mRNA expression level of BD1was significantly down-regulated while the expression level of BD2was significantly upregulated after thermal stress, indicating that the BD2has a direct relationship with B. dorsalis thermal stress, and BD1may play a role in other physical activities.The full length cDNA of Hsp60(GenBank accession number JF812645) was2042bp, with an open reading frame of1722bp, and encoding573amino acids. It contains a molecular Chaperonins cpn60signature sequence AAVEEGIVPGGG, the Apical domain, the Equatorial domain, and the Intermediate domain. Two ATP/ADP binding sites, one Mg2+binding points and a substrate binding site were found. It also had the unique (GGM)n repeat region of the Hsp60family in the C-terminus. The phylogenetic tree result showed that the Hsp60was clustered with Drosophila (Diptera). The Hsp60mRNA expression level was significantly increased after thermal stress, indicating that it could be involved in the B. dorsalis thermal stress response and play an important role in thermal stress.4. Molecular cloning, sequence analysis and mRNA expression profiles of B. dorsalis antioxidant enzyme genesBased on the transcriptome information of B. dorsalis, one full-length cDNA sequence of peroxidase (POD) was cloned, named as BdpoxAl (GenBank accession number JN003585). The full-length cDNA of BdpoxAl is2549bp, with an open reading frame of2106bp, and encoding701amino acids. BdpoxAl has an active-site, an transition state stabilizer, six metal (calcium) characteristics sites and four disulfide characteristic sequence region. Phylogenetic tree indicated that BdpoxAl firstly clustered into one with Nasonia vitripennis, and then gathered together with Culex quinquefasciatus, which implied during the evolutionary process of N. vitripennis and its host Calliphoridae insects, the genes were divergent evolution which is more conducive to parasitic of Calliphoridae insects and lead to this type of developmental genetic relationship. The mRNA expression level of BdpoxA1showed consistent results with enzyme activity determination. The expression level of BdpoxAl were slightly reduced in all treatments except the up-regulation in35℃, indicating that peroxidase encoded by BdpoxA1is a constitutive protein, which could stably decompose H2O2of B. dorsalis regardless of under adversity or normal conditions. Full-length cDNA sequence of a superoxide dismutase (SOD), Bdsodl (GenBank accession number JQ713828) was cloned using the same method. It was946bp with an open reading frame of807bp, and encoding268amino acids, with a Cu/Zn SOD signature (GNAGERIACGII) and six active sites. The phylogenetic analysis showed that the Bdsodl gathered together with Mediterranean fruit fly, which is consistent with the traditional classification. The mRNA expression profiles of Bdsodl suggested that high temperature stress successfully induced the expression of the Bdsodl. The increase of the mRNA expression level of Bdsodl, led to the increased enzyme activity, indicating that more SOD catalyzed disproportionation reaction of superoxide anion, which fully reflects the importance of B. dorsalis SOD to cope with environment thermal stress response.In summary, this study focused on the tight relationship between the organism and their environment temperature changes. The researches were particularly conducted on the important quarantine fruit and vegetable pest of B. dorsalis, from the level of physiology, biochemistry and molecular biology, which led to a comprehensive, systematic study of the impact of extreme environmental temperature on B. dorsalis and their molecular resistance response mechanisms to environmental thermal stress. Firstly, it was studied on the activities of B. dorsalis antioxidant enzymes system in the thermal stress, obtained an overview of the mechanism of antioxidant enzymes to cope with thermal stress. Secondly, the optimized B. dorsalis two-dimensional electrophoresis system was established successfully, obtained a clear pattern of the proteins change under the thermal stress, and identified the major response proteins by the difference in proteomics research. Finally, based on results of the above study, the full-length cDNA sequences of four Hsps and two antioxidant enzyme genes were obtained, and then their expression profile at the mRNA level under thermal stress were investigated, revealed the resistance mechanism in the thermal stress response of B. dorsalis using quantitative real-time qPCR technologies. In conclusion, applying the cutting-edge technologies to research on insect physiology, biochemistry and molecular biology, especially, the application of differential proteomics technology based on the protein level which is the implementation in the physiological functions, supplied a comprehensive analysis of the B. dorsalis to understand the mechanism of resistance on thermal stress, and provided the foundation for comprehensive research on functional genomics and proteomics in more depth, and sole provided some theoretical basis of practical guidance value for field control of B. dorsalis.