关键词:  中华按蚊, 电转化显微注射, 增强型绿色荧光蛋白基因, 芳香烷基胺-N-乙酰基转移酶基因, 瞬时过表达, 基因功能

Abstract: 【Aim】 To construct the electroporation-mediated microinjection platform for Anopheles sinensis and to accomplish the transient overexpression of genes in vivo with the platform for its verification, so as to lay a foundation for the systematic analysis of gene function. 【Methods】 We constructed an electroporation-mediated microinjection platform for An. sinensis using CUY21EDITII electroporator as the main body. The individuals of An. sinensis at 3 h after pupation were injected with transiently overexpressed plasmids containing EGFP and Bm-iAANAT constructed by using in-fusion cloning, and then subjected to electronic shock. The cuticle pigmentation and fluorescence of surviving individuals at 39 h after pupation were observed under stereofluorescence microscopy. The expression levels of EGFP and Bm-iAANAT were detected by qRT-PCR. 【Results】 Overexpression plasmids PIZ-modi-Aepub-EGFP-SV40 and PIZ-modi-Aepub-AANAT-T2A-EGFP-SV40 were constructed and used for microinjection. After injection with PIZ-modi-Aepub-EGFP-SV40 and electric shock, the percentage of surviving melanized pupae of An. sinensis reached 87.5% at 39 h after pupation, of which 92.7% individuals were significantly blocked from melanization and showed obvious green fluorescence compared with the individuals injected with PIZ-modi-EGFP-SV40 without promoter in the negative control group and those injected with overexpression plasmid PIZ-modi-Aepub-EGFP-SV40 without electric shock in the positive control group. In addition, EGFP gene was apparently overexpressed in the fluorescent individuals of the treatment group. The survival rate of individuals after injection with PIZ-modi-Aepub-AANAT-T2A-EGFP-SV40 was up to 80.4% at 39 h after pupation, of which 92.2% individuals were significantly blocked from melanization and appeared obvious green fluorescence compared with the individuals injected with PIZ-modi-AANAT-T2A-EGFP-SV40 without promoter in the negative control group and those injected with overexpression plasmid PIZ-modi-Aepub-AANAT-T2A-EGFP-SV40 without electric shock in the positive control group. Furthermore, the expression levels of Bm-iAANAT and EGFP genes were evidently enhanced in these individuals as well. 【Conclusion】 We successfully constructed the electroporation-mediated microinjection platform for A. sinensis, with which we accomplished the transient overexpression of reporter genes in live pupae conveniently, rapidly and efficiently and produced the target phenotypes, laying a foundation for the functional genome research in A. sinensis.

Key words: Anopheles sinensis, electroporation-mediated microinjection, enhanced green fluorescent protein gene, arylalkylamine-N-acetyltransferase gene, transient overexpression, gene function