Enrichment Of Polyunsaturated Fatty Acids In Musca Domestica Larvae And Preparation Of Fat1 Transgenic Musca Domestica

Objective: To elucidate the enrichment and metabolic transformation of various polyunsaturated fatty acids in food by Musca domestica larvae,and to explore the effects of various polyunsaturated fatty acids on the growth of M.domestica larvae.On this basis,a suitable transgenic method and technology system for M.domestica were established,which laid a foundation for the endogenous synthesis of polyunsaturated fatty acids in M.domestica.Methods: Adding different concentrations(3%,6% and 12%)of polyunsaturated fatty acids(linoleic acid,alpha-linolenic acid,arachidonic acid and docosahexaenoic acid)to the basic diet to feed the M.domestica larvae which had been defatted and subcultured for generations,the total fatty acids of M.domestica larvae were extracted,detected and analyzed by gas chromatography,and the weight of the larvae was noted to analyze the effect of polyunsaturated fatty acids on the growth of M.domestica larvae.The expression vector of Fat1 gene was constructed by using piggyBac transposon vector system.The target gene was introduced into the fertilized eggs of M.domestica by microinjection and electroporation.The positive individuals were screened by detecting the expression of green fluorescent protein after the development of adult flies,and then the tissue DNA was extracted and the Fat1 gene was identified at the molecular level by PCR to establish the technical system of transgenic M.domestica.Results: Linoleic acid,alpha-linolenic acid and arachidonic acid were enriched in M.domestica larvae,and their enrichment degree increased with the increase of the concentration of polyunsaturated fatty acids in food.The highest proportion(the percentage of total fatty acids)of linoleic acid,alpha-linolenic acid and arachidonic acid in larvae were 21.93%,16.13% and 9.68% respectively,while docosahexaenoic acid could not be enriched in M.domestica larvae.The polyunsaturated fatty acids added to the food of M.domestica have been metabolized without the production of new fatty acids in their bodies,while docosahexaenoic acid from food sources were eliminated after catabolism in the larvae of the M.domestica.As for the effect of different concentrations of polyunsaturated fatty acids on the body weight of M.domestica larvae,the body weight of M.domestica larvae increased significantly after feeding alpha-linolenic acid and arachidonic acid,and the larvae weight of the 6% concentrations added group was significantly higher than the other three groups,the larvae of the arachidonic acid addition group at concentrations of 3% and 6% were significantly higher than the other two groups.Using recombinant DNA technology,a plasmid pBac[3×P3]-EGFP-PromA4-Fat1-SV40 polyA was constructed,which can be used to transform M.domestica.In the experiment of transgene preparation by microinjection,one of the G0 generation positive individuals expressed green fluorescent protein was finally obtained.The enhanced green fluorescence was observed in the compound eye and slight fluorescence was observed in the abdomen.The results of PCR showed that the target gene Fat1 could be detected in the head,chest,abdomen,wing and foot of the M.domestica,indicating that the target gene Fat1 had been successfully introduced into the M.domestica.After introducing the transgenic vector by electroporation,three M.domestica were finally able to observe the expression of green fluorescent protein in their body.However,compared with the transgenic M.domestica obtained by microinjection,the expression of green fluorescent protein in their parts was weaker.After extracting DNA from tissues and detected by PCR,The results showed that the amplified bands were rather dim.It was not clear whether the target gene was successfully introduced.Conclusion: The larvae of M.domestica can enrich part of polyunsaturated fatty acids from food.The longer the carbon chain of polyunsaturated fatty acids,the lower the degree of polyunsaturated fatty acids enrichment until they can not be enriched.And the enriched polyunsaturated fatty acids have different effects on the growth of M.domestica larvae.The fact that M.domestica can enrich polyunsaturated fatty acids from food lays a theoretical foundation for the endogenous synthesis of polyunsaturated fatty acids by transgenic M.domestica.Using piggyBac transposon vector system,Fat1 gene encoding polyunsaturated fatty acid desaturase can be introduced into M.domestica fertilized egg and developed into adult ones by microinjection,but the success rate is very low,so it is necessary to continuously improve the microinjection operation technology.However,the success of electroporation of Fat1 gene is still uncertain.It is necessary to further explore the parameters and methods of electroporation.