Baohua Xu

Molecular cloning and characterization of a thioredoxin peroxidase gene from Apis cerana cerana.

Thioredoxin peroxidases (Tpxs) play important roles in protecting organisms against the toxicity of reactive oxygen species (ROS) and regulating intracellular signal transduction. In the present study, we cloned the full cDNA of Tpx1 encoding a 195‐amino acid protein from Apis cerana cerana (Acc). Based on the genomic DNA sequence, a 1442‐bp 5′‐flanking region was obtained, and the putative transcription factor binding sites were predicted. Quantitative PCR analysis showed that AccTpx1 was highly expressed in thorax and that the AccTpx1 transcript reached its highest level in two‐week‐old adult worker honeybees. Moreover, expression of the AccTpx1 transcript was increased by various abiotic stresses, such as ultraviolet light, HgCl2, and insecticide treatments. In addition, the recombinant AccTpx1 protein exhibited antioxidant activity; it removed hydrogen peroxide and protected DNA. These results suggest that AccTpx1 plays an important role in protecting honeybees from oxidative injury and may act in extending the lifespan of them.

Identification, genomic organization, and oxidative stress response of a sigma class glutathione S-transferase gene (AccGSTS1) in the honey bee, Apis cerana cerana

Glutathione S-transferases (GSTs) are members of a multifunctional antioxidant enzyme superfamily that play pivotal roles in both detoxification and protection against oxidative damage caused by reactive oxygen species. In this study, a complementary DNA (cDNA) encoding a sigma class GST was identified in the Chinese honey bee, Apis cerana cerana (AccGSTS1). AccGSTS1 was constitutively expressed in all tissues of adult worker bees, including the brain, fat body, epidermis, muscle, and midgut, with particularly robust transcription in the fat body. Relative messenger RNA expression levels of AccGSTS1 at different developmental stages varied, with the highest levels of expression observed in adults. The potential function of AccGSTS1 in cellular defenses against abiotic stresses (cold, heat, UV, H2O2, HgCl2, and insecticides) was investigated. AccGSTS1 was significantly upregulated in response to all of the treatment conditions examined, although the induction levels were varied. Recombinant AccGSTS1 protein showed characteristic glutathione-conjugating catalytic activity toward 1-chloro-2,4-dinitrobenzene. Functional assays revealed that AccGSTS1 could remove H2O2, thereby protecting DNA from oxidative damage. Escherichia coli overexpressing AccGSTS1 showed long-term resistance under conditions of oxidative stress. Together, these results suggest that AccGSTS1 is a crucial antioxidant enzyme involved in cellular antioxidant defenses and honey bee survival.

A novel Omega-class glutathione S-transferase gene in Apis cerana cerana: molecular characterisation of GSTO2 and its protective effects in oxidative stress

Oxidative stress may be the most significant threat to the survival of living organisms. Glutathione S-transferases (GSTs) serve as the primary defences against xenobiotic and peroxidative-induced oxidative damage. In contrast to other well-defined GST classes, the Omega-class members are poorly understood, particularly in insects. Here, we isolated and characterised the GSTO2 gene from Apis cerana cerana (AccGSTO2). The predicted transcription factor binding sites in the AccGSTO2 promoter suggested possible functions in early development and antioxidant defence. Real-time quantitative PCR (qPCR) and western blot analyses indicated that AccGSTO2 was highly expressed in larvae and was predominantly localised to the brain tissue in adults. Moreover, AccGSTO2 transcription was induced by various abiotic stresses. The purified recombinant AccGSTO2 exhibited glutathione-dependent dehydroascorbate reductase and peroxidase activities. Furthermore, it could prevent DNA damage. In addition, Escherichia coli overexpressing AccGSTO2 displayed resistance to long-term oxidative stress exposure in disc diffusion assays. Taken together, these results suggest that AccGSTO2 plays a protective role in counteracting oxidative stress.

Glutaredoxin 1, glutaredoxin 2, thioredoxin 1, and thioredoxin peroxidase 3 play important roles in antioxidant defense in Apis cerana cerana

Glutaredoxins (Grxs) and thioredoxins (Trxs) play important roles in maintaining intracellular thiol-redox homeostasis by scavenging reactive oxygen species. However, few Grxs and Trxs have been functionally characterized in Apis cerana cerana. In this study, we identified three genes, AccGrx1, AccGrx2, and AccTrx1, and investigated their connection to antioxidant defense. AccGrx1 and AccGrx2 were mainly detected in dark-eyed pupae, whereas AccTrx1 was highly concentrated in 15-day postemergence adults. The expression levels of AccGrx1 and AccTrx1 were the highest in fat body and epidermis, respectively. However, the expression level of AccGrx2 was the highest in muscle, followed by the epidermis. AccGrx1, AccGrx2, and AccTrx1 were induced by 4, 16, and 42°C; H2O2; and pesticide (acaricide, paraquat, cyhalothrin, and phoxime) treatments and repressed by UV light. AccGrx1 and AccGrx2 were upregulated by HgCl2 treatment, whereas AccTrx1 was downregulated. We investigated the knockdown of AccGrx1, AccGrx2, AccTpx-3, and AccTrx1 in A. cerana cerana and surprisingly found that knockdown of the these four genes enhanced the enzymatic activities of CAT and POD; the metabolite contents of hydrogen peroxide, carbonyls, and ascorbate; and the ratios of GSH/GSSG and NADP(+)/NADPH. In addition, we also analyzed the transcripts of other antioxidant genes and found that some were upregulated and others were downregulated, revealing that the upregulated genes may be involved in compensating for the knockdown of AccGrx1, AccGrx2, AccTpx-3, and AccTrx1. Taken together, these results suggest that AccGrx1, AccGrx2, AccTpx-3, and AccTrx1 may play critical roles in antioxidant defense.

Characterization and mutational analysis of omega-class GST (GSTO1) from Apis cerana cerana, a gene involved in response to oxidative stress.

The Omega-class of GSTs (GSTOs) is a class of cytosolic GSTs that have specific structural and functional characteristics that differ from those of other GST groups. In this study, we demonstrated the involvement of the GSTO1 gene from A. cerana cerana in the oxidative stress response and further investigated the effects of three cysteine residues of GSTO1 protein on this response. Real-time quantitative PCR (qPCR) showed that AccGSTO1 was highly expressed in larvae and foragers, primarily in the midgut, epidermis, and flight muscles. The AccGSTO1 mRNA was significantly induced by cold and heat at 1 h and 3 h. The TBA (2-Thiobarbituric acid) method indicated that cold or heat resulted in MDA accumulation, but silencing of AccGSTO1 by RNAi in honeybees increased the concentration of MDA. RNAi also increased the temperature sensitivity of honeybees and markedly reduced their survival. Disc diffusion assay indicated that overexpression of AccGSTO1 in E. coli caused the resistance to long-term oxidative stress. Furthermore, AccGSTO1 was active in an in vitro DNA protection assay. Mutations in Cys-28, Cys-70, and Cys-124 affected the catalytic activity and antioxidant activity of AccGSTO1. The predicted three-dimensional structure of AccGSTO1 was also influenced by the replacement of these cysteine residues. These findings suggest that AccGSTO1 plays a protective role in the response to oxidative stress.

The identification and oxidative stress response of a zeta class glutathione S-transferase (GSTZ1) gene from Apis cerana cerana

Glutathione-S-transferases (GSTs) play an important role in protecting organisms against the toxicity of reactive oxygen species (ROS). However, no information is available for GSTs in the Chinese honey bee (Apis cerana cerana). In this study, we isolated and characterized a zeta class GST gene (AccGSTZ1) from the Chinese honey bee. This gene is present in a single copy and harbors five exons. The deduced amino acid sequence of AccGSTZ1 shared high sequence identity with homologous proteins and contained the highly conserved features of this gene family. The temporal and spatial expression profiles of AccGSTZ1 showed that AccGSTZ1 was highly expressed in fourth instar larvae during development, and the mRNA level of AccGSTZ1 was higher in the epidermis than that in other tissues. The expression pattern under oxidative stress revealed that AccGSTZ1 transcription was significantly upregulated by external factors, such as temperature challenges and H(2)O(2) treatment. The characterization of the purified protein revealed that AccGSTZ1 had low glutathione-conjugating activity, but the recombinant AccGSTZ1 protein displayed high antioxidant activity under oxidative stress. These data suggest that AccGSTZ1 is an oxidative stress-inducible antioxidant enzyme that plays an important role in the protection against oxidative stress and may be of critical importance for the survival of the honey bees.

Molecular cloning, expression and antioxidant characterisation of a typical thioredoxin gene (AccTrx2) in Apis cerana cerana.

Thioredoxins (Trxs) are a family of small, highly conserved and ubiquitous proteins that are involved in protecting organisms against toxic reactive oxygen species (ROS). In this study, a typical thioredoxin 2 gene was isolated from Apis cerana cerana, AccTrx2. The full-length cDNA sequence of AccTrx2 was composed of 407 bp containing a 318 bp open reading frame (ORF) that encodes a predicted protein of 105 amino acids, 11.974 kDa and an isoelectric point of 4.45. Expression profile of AccTrx2 as determined by a quantitative real-time PCR (qRT-PCR) analysis was higher in brain than in other tissues, with its highest transcript occurring on the 15day post-emergence adult and upregulated by such abiotic stresses as 4 °C, 16 °C, 25 °C, H2O2, cyhalothrin, acaricide, paraquat, phoxime and mercury (HgCl2) treatments. However, AccTrx2 was slightly repressed when exposed to 42 °C treatment. Characterisation of the recombinant protein showed that the purified AccTrx2 had insulin disulfide reductase activity and could protect DNA from ROS damage. These results indicate that AccTrx2 functions as an antioxidant that plays an important role in response to oxidative stress.

Identification and characterization of two phospholipid hydroperoxide glutathione peroxidase genes from Apis cerana cerana

Phospholipid hydroperoxide glutathione peroxidase (PHGPX) plays a crucial role in maintaining the integrity of membrane by reducing hydroperoxides of phospholipids. Here, we report the identification and characterization of two genes, designated AccGtpx-1 and AccGtpx-2, encoding PHGPX proteins from the Chinese honeybees, Apis cerana cerana. Alignment analysis showed that AccGtpx-1 and AccGtpx-2 shared high similarity with other known PHGPXs, which show similar structure to thioredoxin. These single copy genes showed complex exon-intron structures. The mRNA of AccGtpx-1 was detected in larvae, pupae and adults and that AccGtpx-2 was only found in adult worker bees. Furthermore, the expression of AccGtpx-1 could be induced by H(2)O(2), ultraviolet (UV) light, heat shock (37 degrees C), HgCl(2), imidacloprid, cyhalothrin, pyriproxyfen and methomyl. In contrast, AccGtpx-2 expression could only be induced by UV. These results indicated for the first time that the AccGtpx-1 and AccGtpx-2 genes encoding A. cerana cerana PHGPXs are regulated differently in response to environmental stressors.

Comparison of the nutrient composition of royal jelly and worker jelly of honey bees ( Apis mellifera )

In this study, the chemical and mineral composition and trace elements in royal jelly (RJ) and worker jelly (WJ) and in royal jelly on particular days (only-2-day RJ [O2d], only-3-day RJ [O3d] and only-4-day RJ [O4d]) were determined. Significant differences in levels of moisture, protein, 10-hydroxy-2-decenoic acid (10-HDA), fructose (F) and glucose (G) were found between the RJ and WJ samples. The nutrient content was significantly higher in samples on O2d than the O3d and O4d samples. The results of this study add to the current knowledge of the nutritional value of RJ and WJ. These results also imply a strong relationship between nutritional effects and polyphenism in honey bees.

Identification and characterization of an Apis cerana cerana Delta class glutathione S-transferase gene (AccGSTD) in response to thermal stress

Glutathione S-transferases (GSTs) are members of a multifunctional enzyme super family that plays a pivotal role in both insecticide resistance and protection against oxidative stress. In this study, we identified a single-copy gene, AccGSTD, as being a Delta class GST in the Chinese honey bee (Apis cerana cerana). A predicted antioxidant response element, CREB, was found in the 1,492-bp 5′-flanking region, suggesting that AccGSTD may be involved in oxidative stress response pathways. Real-time PCR and immunolocalization studies demonstrated that AccGSTD exhibited both developmental- and tissue-specific expression patterns. During development, AccGSTD transcript was increased in adults. The AccGSTD expression level was the highest in the honey bee brain. Thermal stress experiments demonstrated that AccGSTD could be significantly upregulated by temperature changes in a time-dependent manner. It is hypothesized that high expression levels might be due to the increased levels of oxidative stress caused by the temperature challenges. Additionally, functional assays of the recombinant AccGSTD protein revealed that AccGSTD has the capability to protect DNA from oxidative damage. Taken together, these data suggest that AccGSTD may be responsible for antioxidant defense in adult honey bees.