Simulated Knocking Out Developmental Genes Of Honeybee At Egg Stage

Genome editing technique is an advanced technology, which has been used in genetic modification of many organisms in the field of life science. The research in bee genomics is still at the beginning stage, the technique’s application in bee developmental biology should be a way to modify targeted genetic information in living bee. In this study, early embryos of honeybee (Apis mellifera) were used as experimental materials, some development genes at embryonic stage were selected as target genes and their TALENs or CRISPR/Cas9 vectors were constructed respectively before microinjection into embryos in order to study the knockout of genes and thereafter induced mutation, The main results were summarized as follows:1. Four types of transporting container were designed for screening an optimal one on the basis of a maximum hatching rate of eggs transferred out from beehive when they were 5-6h old, because the apiary is more than 16 km far away from the laboratory. Results showed that an average hatching rate as high as 53.64% of eggs was that on the comb foundation coated with halocarbon oil which sustained moisture and carried dissolved oxygen for egg to breath during transportation in a heat preservation box. In a 35℃ thermostatic water bath, eggs couldn’t hatch until 90-100h after oviposition.2. Some parameters of microinjection in a best suitable container were discussed for the purpose of reducing egg damage caused by injection. The results were as follows:in petri dish, eggs on the comb foundation with the middle part-cut down and with a halocarbon oil coating at the nick had a higher hatching rate after microinjection, and the injection efficiency increased with the movement of injective point between tail and back of abdomen. Injecting into the yolk or into the periplasm had little significant influence on the hatching rate of eggs, but injecting into periplasm could improve injection efficiency as its not easy to jam needle tip and had indetectable wound. The needle could easily inject into eggs in a slanting direction with a less spilled contents after its withdrawing, which improved the hatching rate. The hatching rate was relatively stable after injected with 0-2nl of exogenous substances, if more than that volume the hatching rate would decrease, but if less than that volume the functional efficiency would reduce. The developmental duration of eggs after microinjection could be extended to 100-120h in a 35 ℃ thermostatic water bath. There were 23% of eggs to have hatched into larvae among the eggs injected into paraffin oil by the above parameters and incubated for about 100h, with the oil moving with the larval peristalsis in a certain area under the microscope. Dot fluorescence was observed on the eggs after injecting pEGFP-N1 plasmid for 36-48h, but the eggs were so weak that the surviving after 60h were unable to hatch into larvae.3. In order to expediently inspect the result of gene knockout, a method of quick extraction to honeybee genome DNA was discussed. The powder of eggs, larvae or adults were cleaved fully in a 50 mM of NaOH solution at 95 ℃, the genomic DNA was isolated and extracted in Tris-HCl (pH 8.0) at room temperature. The honeybee genome DNA extracted by this method could be used as the template for PCR, the resulting PCR products could be digested completely by restriction enzymes and be used in bee biological experiments with fragment length less than 1500bp.4. A pair of TALEN targeting sites were designed for the developmental genes En, Prd and Tll, respectively, the left and the right TALENs of each gene was synthesized by the unit assembly method, then the mRNA of each gene’s TALEN was transcripted in vitro, which was injected into bee embryos at 5-6h after concentration measurement. After these eggs were incubated at a constant temperature of 35℃ for 48-60h, their gDNAs were tested by restriction enzyme, respectively. The results showed there existed a potential mutation in PCR fragments of gene Tll, a single base mutation was found by sequencing after TA cloning, indicating that TALEN should induce a mutation in a related gene of bee.5. CRISPR/Cas9 targeting sites for the developmental genes Dfd, Eve, Otd-1, Prd and Tll were designed, after the main area around the site was matched to the transcription requirement of T7 promoter in vitro, the targeted sequences were determined and gRNA templates were prepared, the transcriptive for Cas9/gRNA in vitro was purified, and then a mixture of Cas9/gRNA and Cas9 mRNA was injected into bee embryos at 5-6h after the concentration determination, After these eggs were incubated at a constant temperature of 35℃ for 48-60h, their gDNAs were tested by restriction enzyme, respectively. A mutation induced by this technology was found in gene Otd-1 through sequencing TA cloning, showing the technology should edit gene in bee genome.