Identification Of Key Factors Of Honeybee Caste Differentiation

Honeybees are social insects.Under the collective effect of nutrition,hormone,gene,protein,epigenetic and other factors,female honeybees differentiate into queen and worker gradually,and perform different social functions in this social group of honeybees.At present,the caste differentiation mechanism of female honeybees has not been understood fully.In order to further explore the mechanism of honeybee caste differentiation,we collected lymph of queen larvae and worker larvae at 72 h,84 h,96 h and 120 h of the larval stage of Apis mellifera for identifying metabolites and analyzing metabolic pathways,which found out many different metabolites that may be related to honeybee caste differentiation.Among them,439 kinds of metabolites were found in queen honeybees at 72 h,84 h,96 h and 120 h of larval stage,which were different from worker bees,and these metabolites were mainly concentrated in 34 metabolic pathways.There are 487 different metabolites in 84 h,96 h and 120 h of queen honeybee larvaes compared with 72 h,and these different metabolites are mainly concentrated in 24 metabolic pathways.There were 540 different metabolites in 84 h,96 h and 120 h of worker honeybee larvaes compared with 72 h,and these different metabolites are mainly concentrated in 52 metabolic pathways.According to previous research,we analyzed 880 transcription factors in honeybees and found 501 transcription factors were expressed differently between queen larvae and worker larvae.Of these transcription factors,348 were up-regulated only in queen larvae,90 were up-regulated only in worker bee larvae,and 63 were up-regulated both in queen larvae at a certain point in time and in worker larvae another time.In this study,quantitative real-time PCR was used to analyze the expression of five genes AmE75,AmBrc,AmEHMT AmFTZ and AmHR3 in the queen and worker larvae of Apis mellifera for 48 h,84 h and 120 h.The results indicated that the AmE75 gene was significantly higher in queen larvae than in worker larvae at 84 hours of larval stage(p<0.05).AmBrc gene was significantly higher in queen larvae than in worker larvae at 84 h of larval stage(p<0.05)and at the larval stage of 120 h,queen larvae were significantly lower than worker larvae(p<0.05).When AmEHMT gene and AmFTZ gene were in larval stage 84 h and 120 h,queen larvae were significantly lower than worker bee larvae(p<0.05).AmHR3 gene was significantly higher in queen larvae than in worker larvae at 48 h of larval stage(p<0.05)and at 84 h of larval stage,queen larvae were significantly lower than worker bee larvae(p<0.05).Studies by scholars from home and abroad have shown that female honeybee caste differentiation is related to the level of DNA methylation regulated by Amdnmt3 gene.In this study,Apis mellifera larvae cDNA was used as template to clone Apis mellifera Amsp4 gene.The mRNA length of the gene is 2614nt,encoding 831 aa,and consists of 12 exons and 11 introns.Domain analysis showed that the C-terminal of Amsp4 protein has 3 C2H2 zinc finger domains.Fluorescence quantitative PCR was used to analyze the relative expression of Amdnmt3 gene and Amsp4 gene in queen and worker bee at 48 h,84 h and 120 h in larval stage.The results indicated that the expression levels of Amdnmt3 gene and Amsp4 gene were significantly lower in queen larvae at 48h and 84h of larval stage than in worker larvae(P<0.05),and there was no significant difference between queen larvae and worker larvae at 120 h of larval stage(P>0.05).In this study,injecting siRNA of Amsp4 gene into the 60-hour-old worker larvae of Apis mellifera significantly reduced the relative expression of Amsp4 gene and Amdnmt3 gene of worker larvae,and increased the birth weight body length and third backboard length of emerging female honeybees.In order to explore the transcriptional regulation mechanism of AmdnmtS gene.In this study,through 5’RACE experiment we found that Amdnmt3 gene has multiple transcription initiation sites and identified 5 different selective cutting forms.The double luciferase promoter transcriptional activity of the promoter with the highest expression level was detected,and the results show that the promoter has very high transcriptional activity.Sequence analysis of the promoter revealed a number of mammalian SP1/SP3 Gene combination sites.Therefore,we speculate that the mammalian SP1/SP3 protein’s homologue Amsp4 in Apis mellifera may bind to the promoter region of Amdnmt3 gene to regulate its transcription.This thesis is the first time to discover that the Amsp4 gene may regulate the development of female honeybees by regulating the transcription of Amdnmt3 gene.