The Molecular Mechanism Of Transcriptional Regulation Of Vitellogenin Gene In Bombus Lantschouensis(Hymenoptera:Apidae)

During reproduction,vitellogenin(Vg),as an egg yolk precursor,is critical in sexually mature females of oviparous species including insects.The transcription of Vg is usually mediated by hormones such as juvenile hormone(JH),ecdysteroids and neuropeptides.The research about the transcription regulation of insect Vg is mostly focused on Diptera and Lepidoptera.However,little is known in Hymenopteran insects.As pollinator,bumblebees are important for agriculture and ecological systems.Reproducing more offspring workers is necessary for satisfying the pollination in practice.Based on this,it is necessary to decipher the transcriptional profiles and regulation mechanism of the Vg gene in Bombus lantschouensis.The main results include:1.The genomic structure of BlVg and expression levels under different reproductive statusesThe BlVg gene structure(GenBank accession no.MF632304)contained seven exons and six introns,with a length of 7927 bp nucleotides.The BlVg cDNA(GenBank accession no.MF632303)contained 5319 bp nucleotides,encoding a 1772-amino-acids protein with a theoretical isoelectric point(p1)of 6.21 and a calculated molecular weight(MW)of 201.5 kDa.BlVg possessed all the typical vitellogenin domains,including Vitellogenin N,DUF1943,and VWD at the C-terminus.Phylogenetic tree construction revealed that the BlVg protein is most closely related to B.hypocrita Vg.BlVg transcripts were expressed in all the tissues tested.As expected,the highest expression of BlVg transcripts was observed in the fat bodies(383.9-fold for virgin and 3357.07-fold for reproductive queens),followed by flight muscle,head,and ovaries(p<0.05).Additionally,reproductive queens exhibited a significantly higher level than virgins in each tissue(p<0.05).2.The characterization and protein expression of transcription factors Broad-Complex in Bombus lantschouensisFour alternatively spliced BrC isoforms(Z1,Z2,Z3 and Z4)were identified and the full lengths of their CDS were 1491bp,1293bp,1257bp,and 1239bp,respectively.These BlBrC proteins have a common core region,including the BTB domain,and one of four isoform-specific C2H2 zinc finger domains(Z1,Z2,Z3,and Z4)generated by alternative splicing.BIBrC-Z1 and BlBrC-Z3 were expressed obviously higher in ovaries of reproductive queens than those in virgin queens.BlBrC isoforms(Z1,Z2,Z3,and Z4)were expressed successfully in Sf9 cell line.3.The BlVg promoter activity of Bombus lantschouensis with or without hormone inductionMultiple CREs for transcription factor binding were predicted on the BlVg promoter,including TATA box,CAAT box,C/EBP,and DBrC3,DE74 which are ecdysteroid responsive early genes.The promoter activities of distal 2.4 Kb,2.3 Kb,and 1.9 Kb fragments,which contained the two putative ecdysone-related binding sites(DBRC-Z3 and DE74),were significantly enhanced by the addition of 20E but not JH-?.The promoter activity of the 1.4 Kb fragment harboring the DBRC-Z3 binding site was also considerably enhanced by only 20E.The promoter activities of both the Vgl.OK-Luc and Vg0.9K-Luc deletion clones,lacking ecdysone-related binding sites,did not change under ecdysone treatment.These results indicated that the regulation of BlVg transcription can be induced by ecdysone.4.Transcription factor Broad-Complex regulated the BlVg promoter activityThe BlVg promoter activity with the length of 1.9K was strongly enhanced by 20E,while mutation in the BlBrC-Z3 CRE abolished this 20E-mediated activation.In addition,the BlVg promoter activity was markedly increased only upon coexpression with the BlBrC-Z3 isoform.