关键词: 中华按蚊, P2C卵巢导向肽, Gal4蛋白, 12×UAS重复基序, 非胚胎期外源DNA投递技术系统

Abstract: 【Aim】Based on the features that the P2C can be delivered into ovaries and the Gal4 protein can stably bind to the UAS sequence, to establish an efficient non-embryonic exogenous DNA delivery technical system in Anopheles sinensis.【Methods】The recombinant protein P2C-Gal4-DsRed was injected into the abdomen of female adults of A. sinensis at 20 h after sucking blood. The delivery efficiency of the recombinant protein P2C-Gal4-DsRed in the ovaries was analyzed by frozen section fluorescence observation and Western blot. The recombinant P2C-Gal4 DNA BINDING protein was prepared, transgenic plasmid and helper plasmid containing the 12×UAS repeat motif were constructed, and the in vitro binding between the recombinant protein P2C-Gal4 DNA BINDING and 12×UAS repeat motif was analyzed by electrophoretic mobility shift assay. The complexes P2C-Gal4 DNA BINDING recombinant protein+helper plasmid ITF36-12×UAS and P2C-Gal4 DNA BINDING recombinant protein+transgenic plasmid ITF2-12×UAS afm incubated in vitro were injected into the abdomen of female adults of A. sinensis at 20 h after sucking blood, and the DNA of their ovaries was extracted at 40 h after a blood meal. The delivery of exogenous DNA in vivo was analyzed by PCR amplification with specific primers and sequencing. 【Results】The ovaries of 100% female adults of A. sinensis injected with P2C-Gal4-DsRed showed obvious red fluorescence under the green filter, indicating that the P2C-Gal4-DsRed recombinant protein could be efficiently transferred into the ovaries of female adults of A. sinensis. The recombinant P2C-Gal4 DNA BINDING protein could stably bind to the 12×UAS repeat motif and the plasmid containing this repeat motif fragment. Exogenous DNA fragments were detected in the ovarian tissues of 91% and 93% of female adults of A. sinensis injected with P2C-Gal4 DNA BINDINGP2C-Gal4+ITF36-12×UAS and P2C-Gal4 DNA BINDING+ITF2-12×UAS afm, respectively. 【Conclusion】The exogenous DNA delivery technical system based on the P2C ovary-delivering peptide and the Gal4-12×UAS binding property was successfully established in A. sinensis. Through this technology platform, DNA molecules such as plasmids can be conveniently, rapidly and efficiently delivered into the ovaries of A. sinensis, laying a foundation for further simplifying genetic operations such as transgene, overexpression and gene knock-in.

Key words: Anopheles sinensis, P2C ovary-delivering peptide, Gal4 protein, 12×UAS repeat motif, non-embryonic exogenous DNA delivery technical system