Gene Editing Of Sex Determining Genes Fem And Amdsx In Apis Mellifera

Honeybee is a typical social insect.Females are diploid developed from fertilized eggs,while males are haploid developed from unfertilized eggs.The sex of honeybee is regulated by a cascade reaction consisting of csd→fem→Amdsx.The main purpose of this paper is to edit fem and Amdsx gene by CRISPR-Cas9 method,and analyze the transcriptome of diploid embryo fem gene after editing.In this study,the fem gene of western honeybee was edited.The results showed that after the fem gene was knocked out in the diploid embryo stage,the sex changed from female to male after the individuals developed into adults,and the compound eyes of the mutant individuals were obvious,and drone testis and mucous glands were found in the mutant individuals.By detecting the expression of Amdsx gene downstream of fem gene,we found that the female splicing form of Amdsx ~Fof the mutant Amdsx gene almost disappeared,while the male splicing form Amdsx ~Mappeared.In addition,the first generation sequencing and TA cloning analysis also confirmed the occurrence of individual base deletion.Fem gene plays an important role in female development and maintenance.In addition,transcriptome analysis was performed on diploid Western honeybee embryo after fem gene editing,and a total of 362.98M high-quality Reads were obtained after data filtering.The cumulative length of single sample sequence ranges from 6.35GB to 7.27GB.As the number of Clean Reads Q30 was higher than 93.17%,and a counting of Clean Reads Q30 was between 93.41%and 94.06%.Through data analysis,a total of 155differentially expressed genes were screened out.Enrichment analysis of GO and KEGG pathways indicated that insect hormone biosynthesis,terpenoid backbone biosynthesis,phenylalanine,tyrosine and tryptophan biosynthesis were significantly enriched.Besides,genes related to bee sex determination or ovarian development(fem,vitellogenin)were found to be significantly different between the two groups.Finally,the haploid and diploid Amdsx genes of western honeybee were edited using the same gene editing method.An adult was obtained from experiments on haploid embryos.The appearance of the individual is still similar to that of the drone.Through anatomy,we did not find obvious gonads,but there is a queen’s unique spermatheca at the tail of the abdomen of the individual.At the same time,fat bodies were abundant in the abdomen of the individual.The expression of vg gene was quantitatively detected by Q-PCR.We found that the expression of vg gene in the individual was not significantly different from that in the queen,but significantly different from that in the drone.We speculated that the individual had feminization.In addition,we designed sg RNA for the female-specific splicing form of Amdsx,and used the same method to inject sg RNA of Amdsx into diploid embryos.Phenotypes and anatomical tissues of the cultivated adult were observed,and no changes in phenotypes and internal structures were observed.After first-generation sequencing,it was found that,three individuals had base deletions.