Isolation And Characteristic Analysis Of Stress Resistance Related Genes GST, CacyBP, NAChR Subunits From Apis Cerana Cerana

Resistance study of living organisms is of great significance and is currently a researchhotspot. The organism has an elaorate defense system against harmful foreign or endogenoussubstances. The Chinese bees which are the excellent indigenous bee species are of greatsignificance to the maintenance of biological diversity of plant resources in China, and topromote the sustainable development of beekeeping. At the same time, the Chinese bees canserve as a model organism for studying social behavior, learning and memory in navigationbehavior. However, due to the deterioration of the environment, the worker bees in theprocess of metamorphosis from larvae, pupae and adults will encounter a variety of stress, andthere is a crisis of survival. Therefore, in order to better protect and use this unique resourcesin the bees, the physiological function and molecular mechanisms in the bees from themolecular level is imperative. To the end, we separated the genes from differentdevelopmental stages in the bees involved in the resistance responses, and analyzedexpression characteristics. The results are as follows:（1） A GSTs4gene named AccGSTs4was identified from the larvae of A. cerana cerana. Itencoded a203amino acid polypeptide, which exhibited the structural motif and domainorganization characteristic of GST. RT-qPCR indicated that AccGSTs4was presented in allstages of development with high level in4th instar larvae. Immunolocalization furtherrevealed the distribution of AccGSTs4in4th instar larvae. Expression analysis showed thatthe transcripts of AccGSTs4from the larvae were upregulated under dietary HgCl₂. The GSTactivity under stress was higher than the controls fed on HgCl₂-free diet. Disc diffusion assayprovided further evidence of recAccGSTs4resistance to long-term exposure of HgCl₂stress.Additionally, analysis of promoter analysis further clarified the probable expression patternsof AccGSTs4. Taken together, our findings indicate that the larvae AccGSTs4may play a rolein mercury stress response, and it will help to protect honeybees from heavy metals.（2） We cloned and characterized one CacyBP gene, referred to AccCacyBP, from the honeybee （A. cerana cerana）. Bioinformatic analysis indicated that AccCacyBP was highlyconserved and closely related to the CacyBP of other insects. Promoter analysis revealed anumber of putative tissue, development and stress-related transcription factor-binding sites.Moreover, expression analysis demonstrated that AccCacyBP was expressed at all of thestages of development, especially in the brains of honeybees. Immunohistochemistry analysisshowed the existence of AccCacyBP in the brain. The transcript levels of AccCacyBP in thebrains of honeybees were developmentally induced and upregulated by exposure to oxidativestresses, including UV-light, acetamiprid and HgCl₂. This study demonstrates that the CacyBPgene in honeybees may be a neuronal protein involved in the developmental regulation andthe stress-response of the brain of honeybees.（3） We identified and characterized two novel β subunit genes （Accβ1and Accβ2） from A.cerana cerana. Homology analysis indicated that Accβ1and Accβ2possess characteristicsthat are typical of nAChR subunits although Accβ2was distinct from Accβ1and the othernAChR subunits, due to its unusual transmembrane structure and uncommon exon-intronboundary within the genomic region encoding the TM1transmembrane domain. Analysis ofthe promotor sequence indicated that Accβ1and Accβ2possess different regulatory elements,suggesting that the genes might exhibit various expression and regulatory patterns. RT-PCRanalysis demonstrated that Accβ2was expressed at a much higher level than Accβ1in thetissues of adult bees. Moreover, Accβ1was highly expressed at the pupal stages, whereasAccβ2was abundantly expressed at the larval stages. Accβ1and Accβ2were both induced byexposure to various insecticides and environmental stresses although Accβ2was moreresponsive than Accβ1. These results indicate that both Accβ1and Accβ2may have differentroles in insect growth and development. They may belong to separate regulatory pathwaysinvolved in the response to insecticides and environmental stresses.