Proteomics And MicroRNA Analysis Of Female Bumblebees,Bombus Lantschouensis (Hymenoptera,Apidae)

Bombus lantschouensis is a native bumblebee species in China.Due to its excellent pollination performance and potential for the factory reproduction,this species has been reared in the laboratories within China for the pollination services.Unfortunately,many queens of B.lantschouensis can't lay eggs after hibernation which is the crucial factor for the development and establishment of the colony.Hence,during this study,we tried to analyze the certain physiological factors which might be responsible for the egg laying regulation of queens.Using differential proteome technology,we analyzed the differences between haemolymph proteins of female bumble bee species at different reproductive status.Similarly,the total miRNA species and differences between the different reproductive statuses of female bee were also explored using high-throughput sequencing solexa sequencing technology.Finally,two candidate proteins were verified and analyzed.The main results were as follow;1.Proteomics research on haemolymph protein of female bumblebeeThe proteome of haemolymph between the different females of B.lantschouensis under different reproductive status were compared using gel-based(one-dimensional gel electrophoresis,two-dimensional gel electrophoresis)proteomics approaches.The results showed that the total protein concentration of haemolymph can be used as a marker to judge whether female bees were oviparous.The expression profile of both egg-laying and non-egg-laying female bees was obtained using LC-MS/MS.The numbers of identified proteins in egg-laying and non-egg-laying queens and workers were 783,463,275 and 314 respectively.Of the total proteins,128 were co-expressed in female bees and 44 showed the significantly difference in abundance.Gene Ontology analysis used to compare the non-egg-laying queens.The result indicates that the functional composition of haemolymph protein causes some significant changes in reproductive process,behavior,molecular function,cell composition of the protein,in stimulating response as well as in reducing some cellular metabolism Oxidative proteins.2.Analysis of microRNA in ovary of B.lantschouensisA total of 329 known miRNAs were obtained using mirDeep2 for the prediction of miRNA of queen's ovary tissue,of which 75 were mature miRNAs already known to honeybee(Apis mellifera).Additional 255 new mature mi RNAs were obtained using BLAST with the whole genome of the Bombus terrestris.The expression analysis of miRNA indicated that all the miRNAs levels were down-regulated significantly in queens after starting egg layingg.Four different algorithms and one consensus rank approach were employed to analyse the qPCR data.The results show that the candidate reference genes of different reproductive statuses are variable largely across different tissues.For the head and ovary,miR275 was the best candidate.For head,miR275 was the most stable candidate,while the candidate for the ovary was miR277.3.Gene cloning and analysis of IRP30 expression profile from B.lantschouensisThe IRP30 gene from B.lantschouensis was cloned using the RACE method and touch-down PCR.It encodes only one transcript.The full-length of cDNA was 1082 bp.It contains a full open reading frame(ORF)of 825 bp,which codes for 275 aa protein.The signal peptide cleavage site determined by SignalP 4.1 showed that the N-terminus has a signal peptide sequence.Analysis of conserved domains indicated that IRP30 had multiple leucine-rich repeats between 35-180 amino acids.RT-qPCR approach's results showed that expression levels of IRP30 were higher in Pb and Pbl stages than in other stages(P<0.01).The expression levels of IRP30 between three caste of bees were;worker> queen> drone and similarly,between different tissues these were;throax> head> abdomen.The relative expressions of IRP30 in head,muscle and fat body were higher than other tissues.RT-qPCR and Western Blot demonstrated that IRP30 was up-regulated significantly in female under egg-laying(P<0.01).Consequently,we suggest that IRP30 is involved in the reproductive transformation in female bees.4.Gene cloning and analysis of PEBP expression profile from B.lantschouensisThe PEBP gene from B.lantschouensis was also cloned by the RACE method.In contrast of IRP30 gene,it encodes two transcripts;PEBPX1 and PEBPX2.The two full-length fragments of these cDNAs contain 1005 and 915 bp respectively.Both PEBPX1 and PEBPX2 transcripts contain full open reading frames(ORF)of 627 bp and 549 bp which code for 208 aa and 182 aa proteins respectively.The signal peptide cleavage site determined by SignalP 4.1 showed that the cleavage site of the protein product from the PEBPX1 is between positions 29 and 30.However,there was no signal peptide in the predicted protein sequence of PEBPX2.Furthermore,we investigated PEBP transcription levels in workers at different developmental stages by quantitative RT-PCR.Expression levels for both PEBPX1 and PEBPX2 were higher in the egg at Pbl and Pbd stages as compared to other stages(P<0.01).Such findings indicate that PEBP is very important in these developmental stages.The expression level of PEBPX2 was much higher than PEBPX1 in most of the stages except the Pha pupae stages(P<0.01).The expression of transcript PEBPX2 was more preference than PEBPX1.Quantitative PCR and Western Blot demonstrated that PEBP was up-regulated significantly in female under egg-laying.Therefore we suggest that levels of these two PEBPs could be related to the regulation of reproduction in bumblebees.In conclusion,during our current study we identified the differences of protein numbers,types and functions from the female bee's haemolymph between egg-laying and non-egg-laying in B.lantschouensis.The mi RNAs were predicted in queens' ovarian under different reproductive statues.At the same time,the most stable candidate genes were also screened in B.lantschouensis for evaluating expression of miRNA.Lastly,the expressional characteristics of candidate proteins IRP30 and PEBP in the bumblebees were also studied.Our results will be beneficial for the study of molecular biology and reproductive physiology of bumblebee as well as for the improvement of factory rearing technologies of native bumblebees.