Molecular Mechanism Of Honeybee NF-Y Gene Family In Response To Heat Stress

As important special economic animal,honeybees not only provide abundant honeybee products for human,but also play important roles in crop pollination and maintaining terrestrial ecosystems.However,recently,there is clear evidence of severe honeybee declines in the world.It has been demonstrated that different stresses,including heat stress,are among the primary drivers of this decline.Heat stress has deleterious effects on the growth,development and foragering ability of honeybee.Therefore,researching honeybee heat tolerance,improving and breeding new heat resistant honeybee species are an important prerequisite for the healthy development of honeybee colony.Nevertheless,knowledge about detailed molecular mechanism of how honeybees defend against heat stress is still little.Nuclear Factor Y?NF-Y?is one important transcription factors,and consists of three distinct subunits?NF-YA,NF-YB and NF-YC?that exist in almost all organisms.NF-Y has become a hot topic in the field of molecular biology research in recent years.The functions of animal NF-Y have been revealed mainly in growth,development,tumour,cancer and other human diseases.It has been revealed that plant NF-Y plays an important role in heat stress response.However,the molecular mechanism of animal NF-Y in heat stress response is still unclear.Here,we used Apis cerana cerana and Apis mellifera ligustica as experimental material.We first performed an RNA-sequencing?RNA-seq?assay to select heat response genes.The NF-Y gene family was isolated and identified.Then,we used molecular biology and cell biology assays to uncover the underlying regulatory mechanisms of honeybee heat resistance mediated by NF-Y.The main results are as follows:?1?RNA-seq results showed that heat stress induced the transcription of many gene family,including NF-Y.The results from gene function annotation found that heat stress influenced many important biological processes and signaling pathways in honeybee,such as growth,development,immune system and longevity regulating.These results indicate that heat stress response genes,including NF-Y,may play crucial roles in honeybee heat stress response.?2?Bioinformatics analysis indicate that the amino acid sequence homology of NF-YA,NF-YB and NF-YC in Apis cerana cerana and Apis mellifera is 100%.Though the genomic structure of Apis cerana cerana and Apis mellifera NF-Y were obviously different from those in other animals,honeybee NF-Y had typical NF-Y conserved domain.The conserved domain of honeybee NF-YA had two alpha-helices,and the conserved domains of NF-YB and NF-YC had four alpha-helices.We conclude that the conserved domains of NF-Y may be indispensable for the function of animal NF-Y.?3?Quantitative real-time PCR?qRT-PCR?results showed that the expression of NF-YA,NF-YB and NF-YC was all induced under short and long heat stress in Apis cerana cerana and Apis mellifera compared to the control.Moreover,under short-trem heat stress,the upregulation level of NF-YA is higher than that of NF-YB and NF-YC in Apis cerana cerana,and the expression of NF-YA and NF-YB was higher than NF-YC in Apis mellifera.However,the difference of expression levels of NF-YA,NF-YB and NF-YC was not obvious under long-term heat stress.We speculate that the difference of NF-YA,NF-YB and NF-YC expression level is the specific demand of honeybee to NF-YA,NF-YB and NF-YC under difference degree heat stress.To further prove the function of NF-Y under heat stress,we silenced NF-YB and NF-YC using double-stranded RNA mediated RNA interference assay in Apis cerana cerana.We found that silencing NF-YB and NF-YC increased the death rate of Apis cerana cerana under heat stress.These results indicate that NF-Y plays important roles in honeybees under heat stress.?4?Subcellular localization analysis showed that NF-YA presented in the nucleus,NF-YB localized in the cytoplasm,and NF-Y accumulated in both the nucleus and cytoplasm.Yeast two-hybrid result presented that NF-YA interacted with NF-YC,and NF-YB interacted with NF-YC,while there was no interaction between NF-YA and NF-YB.Co-immunoprecipitation?Co-IP?and immunostaining assays further demonstrated this result.NF-YC carries NF-YB from the cytoplasm into the nucleus after interacting with NF-YB.After truncating the protein sequence of NF-YB and NF-YC,we found that histone fold motif?HFM?is indispensable for the interaction of honeybee NF-YB and NF-YC using Co-IP and immunostaining assays.In addition,immunostaining assays results showed that HFM is essential for the coaccumulation of NF-YB and NF-YC in the nucleus.Deletion of HFM from NF-YB and NF-YC caused NF-YB and NF-YC expressing both in cytoplasm.?5?RNA-seq result showed that knockdown of NF-YB and NF-YC influenced many vital biological processes and signaling pathways,including environmental stress response,development,antioxidant defense response and longevity regulating,in Apis cerana cerana.In addition,knockdown of NF-YB and NF-YC had effects on the expression of many heat response genes,especially LOC108003540 and LOC107994062,in Apis cerana cerana.Heat stress induced the expression of these two genes compared to the control.However,knockdown of NF-YB and NF-YC repressed the induction degreee of LOC108003540 and LOC107994062 under heat stress compared to the control in Apis cerana cerana.These results indicate that NF-Y participate in honeybee heat stress response through regulating the expression of heat-induced genes.?6?The expression of NF-YA,NF-YB and NF-YC were induced under oxidant hydrogen peroxide?H₂O₂?treatment compared to the control in Apis cerana cerana.The transcription of NF-YA,NF-YB and NF-YC were inhibited under antioxidant vitamin C?VC?treatment compared to the control in Apis cerana cerana.Silencing NF-YB and NF-YC increased the contents of H₂O₂,malondialdehyde and protein carbonyl,indicating that knockdown of NF-YB and NF-YC increased honeybee oxidative damage degree.Besides,RNA-seq and qRT-PCR results showed that silencing of NF-YB and NF-YC decreased the expression of many antioxidant genes.The enzymatic activity of peroxidase,superoxide dismutase,glutathione S-transferase and catalase,and the content of VC were also decreased after silencing NF-YB and NF-YC in Apis cerana cerana.These results indicate that the antioxidant ability of honeybees may be influenced by NF-Y knockdown.Indeed,we found that knockdown of NF-YB and NF-YC reduced the total antioxidant capacity of honeybees in Apis cerana cerana.These results indicate that knockdown of NF-YB and NF-YC influences the antioxidant signaling pathways,decreases the antioxidant capacity,and increases oxidative damage in honeybees.Collectively,we found that NF-YA,NF-YB and NF-YC played important roles in honeybee heat stress response.Nuclear localization of NF-YB depended on its connection with NF-YC.NF-Y regulated honeybee heat stress response by mediating heat stress response genes and antioxidant defense system.Upregulation of NF-Y contributes to increase honeybee antioxidant capacity,decrease the oxidative damage from heat stress,thus enhancing the survival ability of honeybees under heat stress.