Differential Gene Expression Analysis Between Spermary And Ovary In Apis Mellifera And Function Study Of Ampabp2 And Amrbp1 Gene

The sexes of honeybees are determined by haplo-diploid sex determination system.The males are haploid developed from unfertilized eggs,and the females are diploid developed from fertilized eggs.In order to make clear the molecular mechanism of honeybee sex determination and differentiation,we analyzed the differentially expressed genes between spermary and ovary in Apis mellifera ligustica,and studied the function of Ampabp2 and Amrbp1 in honeybee sex determination pathway.In this study,four transcriptome libraries were constructed,which were two ovarian biological replicates(yc-1,yc-2)and two testis biological replicates(yx-1,yx-2).After removing the joint and the low-quality Data,we obtained 74,672,958 clean reads corresponding to 18.79Gb total nucleotides.The clean reads of each sample ranged from4,443,325,050-5,062,141,958 corresponding to 4.44Gb nucleotides,GC content between40.6%-42.26%,and the Q30 base percentage were above 94.29%.Sequence alignment with the Western honeybee genome,the Uniq Mapped Reads of each sample ranged from26,885,905 to 31,190,223.In this study,a total of 4367 differentially expressed genes were identified.of which 2227 up-regulated and 2140 down-regulated in testis compared to ovaries.Based on the NR,Swiss-Prot,GO,COG,and KEGG public databases with a cut-off E-value of 10~-55 using Blastx,a total of 3604 unigenes were annotated.Knockdown studies showed that the injection of Ampabp2 siRNA induced male splicing of Amdsx mRNAs in females,which is entirely absent in the control embryos,and the female splicing of the Amdsx gene almost disappeared,which produce only the Amdsx female splice product.This result suggests that the Ampabp2 gene is essential to promote female splicing of Amdsx transcripts.Similarly,compared to our control embryos,the male splicing of fem mRNAs in the treatment of female embryos was enhanced,while the female splicing weakened,indicating that the Ampabp2 gene was essential for the female splicing of fem.The result showed both Amdsx and fem in the treatment of female embryos have male splice patterns.Thus,the Ampabp2 gene may be interacted with CSD to regulate the female splicing of the fem gene.In this study,the expression of Amrbp1 gene in different developmental stages of female and male embryos and larvae in western honeybees was analyzed by semi quantitative RT-PCR.The results showed that the three splicing variants of Amrbp1 gene(Amrbp1-R1,-R2,-R3)were expressed in both male and female embryos and larvae at different developmental stages,and there was no male-or female-specific splicing variants.GST pull-down experiment showed that there is no interaction between Amrbp1 and Csd.