Studies On The Technology Of Artificial Feeding Apis Cerana Cerana Fabricius In Laboratory And Transferring ECFP Into Eggs By Microinjection

It is well known that the Apis cerana cerana Fabricius is one of the precious honeybee species. The artificial feeding technology of Apis cerana cerana Fabricius eggs and larvae has not been successful yet. It is the basic work for the research of nutrition requirement, growth and development and transgene of Apis cerana cerana Fabricius to establish the complete technology system of artificial feeding of Apis cerana cerana Fabricius eggs and larvae in laboratory. Basing on this artificial feeding system, transgenic research was carried out to Apis cerana cerana Fabricius by microinjection. This is the basic work of utilizing honeybee as bioreactors to product animal medicinal protein.The Apis cerana cerana Fabricius eggs and larvae were collected as experiment objects in present study. With the aim of normal development of Apis cerana cerana Fabricius eggs and larva in laboratory, this study initially established the technology system of feeding Apis cerana cerana Fabricius in laboratory and simulating the environmental condition of bee larvae development in nature to explore the feasible mode of feeding larvae. The exogenous gene ECFP was transferred into Apis cerana cerana Fabricius eggs to establish transgenic microinjection method of Apis cerana cerana Fabricius eggs by microinjection. Results are as follows:1. By screening we established the optimal dietary nutrition formula of Apis cerana cerana Fabricius larvae. This study provided nutritional parameter for the artificial feeding larvae of Apis cerana cerana Fabricius with large scale.2. The difference of pupation number was significant(p<0.05)between two models of"transferred daily"and"non-transferred". The pupation rate of larvae reared of group with"non-transferred"was higher 12.7 percent than"transferred daily"and the survival rate was higher 9.7 percent. This indicated that the pattern of"non-transferred"is better than"transferred daily".3. It was for the first time that the Apis cerana cerana Fabricius eggs were incubated successfully in laboratory. The 0-6h eggs were incubated (95%R.H,34℃) and the survival rate is 11.1 percent.4. The plasmid vectors pCasper-hs-orf and pXL-BacⅡ-ECFP which contained piggyBac ORF and 3×P3-ECFP were identified by single enzyme digestion, double enzyme digestion and PCR.5. ECFP was transferred into 0-4h Apis cerana cerana Fabricius eggs by microinjection. The treated eggs were cultured by artificial rearing in vitro and reared in a natural colony respectively. The results of fluorescence detection showed that no positive eggs.In conclusion, it was realized that the Apis cerana cerana Fabricius eggs and larvae were incubated successfully in laboratory. The eligible environmental parameter and nutritional parameter were to obtain in this study. It was established that the"non- transferred"pattern in laboratory according to this study.The Apis cerana cerana Fabricius eggs were injected ECFP into by microinjection.But no positive eggs were found. The results showed that the microinjection for the Apis cerana cerana Fabricius eggs needed to be further studied and improved in technical details.