中华蜜蜂电穿孔外源基因表达系统的构建及其应用OA

[Aim]To establish an in vivo electroporation system for exogenous gene expression in Apis cerana cerana,utilize this system to evaluate the transcription-driving efficiency of promoters from different sources within A.c.cerana,then optimize the construction of highly efficient and specific genetic engineering vectors for A.c.cerana.[Methods]Overexpression plasmids carrying the EGFP reporter gene were constructed using homologous recombination.Plasmids were microinjected into the red-eyed pupae of A.c.cerana through the spiracle of the 2nd abdominal sternite,followed by electroporation at the injection site.The EGFP expression-driving efficiency of the endogenous AcActin5c promotor(pAcActin5c)from A.c.cerana,the endogenous AmlActin5c promotor(pAmlActin5c)from A.m.ligustica,and the cytomegalovirus(CMV)promoter(pCMV)after electroporation in A.c.cerana was comprehensively analyzed by integrating microinjection technology,in vivo electroporation technology and reporter gene quantification.[Results]The vectors for overexpression of pAcActin5c-EGFP,pAmlActin5c-EGFP,pCMV-EGFP and EGFP for microinjection were successfully constructed.An in vivo electroporation system for exogenous gene expression applicable to A.c.cerana was established by combining the microinjection system and electrotransformation system.All the three promoters from different sources(pAcActin5c,pAmlActin5c and pCMV)exhibited significant initiation effects on EGFP expression within A.c.cerana after plasmid injection and electroporation.In contrast,no obvious initiation effect on EGFP expression was observed for any of the three promoters in the treatment group that received only expression plasmid injection without electroporation.Furthermore,differences in the initiation timing on EGFP expression among the promoters from different sources were observed.pAcActin5c from A.c.cerana responded relatively quickly,and initiated the expression of the exogenous gene EGFP after electroporation,whereas pAmlActin5c from A.m.ligustica and the viral pCMV responded more slowly.[Conclusion]This study successfully established an in vivo electroporation system for exogenous gene expression in A.c.cerana for the first time.This system can be used to rapidly screen highly efficient promoters suitable for A.c.cerana and provide an important tool for more in-depth gene function research in A.c.cerana.