Analysis Of Differential Expression Genesbetween Male And Female During Embryonic Period In Apis Mellifera And Editing Of Ampabp2 Gene

In honeybees,males are haploid,develop from unfertilized eggs,and females are diploid,develop from fertilized eggs.The purpose of this study was to detect the differentially expressed genes(DEG)between male and female during the embryonic stage and to study the function of Ampabp2 by gene editing.A total of 30 transcriptome libraries were constructed in t his study.Six colonies were selected for t his experiment.Three of them were headed by single male fertilized queens laying fertilized eggs,and anot her three colonies were headed by unfertilized queens laying unfertilized eggs.The time point when he queen has finis hed laying eggs for six hours was regarded as 0 h,and the eggs were collected at 0 h,24 h,36 h,48 h and 72 h.A total of 1331.63 M high-quality reads were obtained after data filtration.The cumulative sequence length of each sample was between 5.96 Gb-6.82 Gb with an average of 6.658 Gb.The Q30 of clean reads was higher than 90.7%,and the uniquely mapping rate is77.11%-87.03%,indicating high data reliability.The DEGs between female and male embryos at different time points were screened out using the RNA-seq data.The results showed that there were a total of 4541 DEGs between female and male embryos at all t he five time points,of which 5 DEGs showed expression difference in all the five time points.In 24 h,36 h,48 h,72 h,there are a total of 4485 differentially expressed genes,37 of them are differentially expressed in all the four time points,of them,34 genes upregulated in female and 3 upregulated in male.The GO and KEGG enric hment analysis of DEGs s howed significant enric hment in lysine degradation,chitin metabolism and cross-model transport pathways.In addition,the key sex determination gene csd and four genes related to gonadal development(vg,squid,vas and ago2)were significantly expressed between male and female embryos.In this study,the Ampabp2 gene of Apis mellifera was edited by CRISPR/Cas9 tec hnology.Since the eggs after injection are different from he eggs bred normally,there are certain requirements for the culture conditions,especially humidity.Therefore,in this study,three different humidity levels were selected to explore the cultivation condition of eggs after injection.The results showed that the mortality rate of the larvae cultured at 85%humidity was low than that at 75% and 95% humidity,the larvae at 75% humidity were dried up due to injury,and the larvae at 95% humidity were become water droplet.Based on the previous experimental results that Ampabp2 interacts with csd,We guess that Ampabp2 genes is involved in honeybee sex determination,therefore,in this study we used CRISPR/Cas9 technology to edit Ampabp2,We found that an individual died in the larval stage had editing of the Ampabp2 gene,and no individual had editing of the Ampabp2 gene in the embryonic and adult stages.We speculate that deletion of Ampabp2 gene CRISPR/Cas9 might be lethal to honeybee embryos.