De Novo Sequencing Of Transcriptome And Gene Expression Analysis Of Females Honeybees In Apis Cerana Cerana

With the Apis cerana cerana as materials in this study, we obtained the transcriptome information of A. cerana cerana by RNA-seq and compared gene expression difference between queens and workers of A.cerana cerana by digital gene expression (DGE) analysis. In additon.cDNA cloning and expression analysis of the A.cerana cerana DNA methyltransferase3(Dnmt3)gene were performed.Using high-throughput illumina RNA sequencing we obtained51.581.510clean reads corresponding to4.64Gb total nucleotides. These reads were assembled into46999uniques with a mean length of676bp. Based on sequence similarity search against the five public databases (NR,Swissport.GO,COG,KEGG) with a cut-off E-value of10"5using Blastx, a total of24.630unigenes were annotated. Using these transcriptome data as reference sequences, we analyzed the gene expression difference between the queens and workers of the A.cerana cerana using a tag-based digital gene expression method, we obtained about5,962,735and5.663,952tags from the queens and workers sample sequencing, respectively. A total of414genes were detected differentially expressed between them,with189up-regulated and225down-regulated in queens compared to workers.We chosed ten differentially expressed unigenes to detect their expression between adult queens and workers using quantitative RT-PCR.The result showed that the real time PCR results of all these genes are consistent with the DGE data.The DNA methyltransferase3(Dnmt3) in A. cerana cerana was cloned by using RT-PCR, and the quantitative analysis of the expression level of Dnmt3mRNA in different developmental stages of worker and queen were conducted using real-time qPCR. The full-length cDNA of Dnmt3gene was2277bp, encoding758amino acids, and the predicted MW and PI were88.24kD and7.85, respectively. The deduced amino acid sequence of Dnmt3showed the highest identity (99%) with that of Apis mellifera. The Dnmt3transcript was clearly detected in different developmental stages of worker and queen, and it was expressed significantly higher in30d-old worker than in1d-and7d-old worker(P<0.05), while no difference existed between1d-and7d-old worker (P>0.05). The Dnmt3transcript was expressed higher in queen pupae than in worker pupae (P<0.05), and was higher in1d-old queen than in1d-old worker (P<0.05). The expression level of Dnmt3between laying worker and laying queen had no significant difference. The results suggested that Dnmt3may be involved in the division of labor in workers and ovary development in honeybees.