中华按蚊中基于卵巢导向肽和Gal4-UAS结合特性的外源DNA投递系统构建

Construction of exogenous DNA delivery system based on ovary-delivering peptide and Gal4-UAS binding property in Anopheles sinensis(Diptera:Culicidae)

【目的】利用P2C可以定向进入卵巢以及Gal4蛋白可与UAS序列稳定结合的特点,在中华按蚊Anopheles sinensis中建立高效的非胚胎期外源DNA投递技术系统。【方法】注射P2C-Gal4-DsRed重组蛋白至吸血后20 h时的中华按蚊雌成蚊腹部,通过冰冻切片荧光观察和Western blot检测分析重组蛋白P2C-Gal4-DsRed在卵巢中的投递效率;制备P2C-Gal4 DNA BINDING重组蛋白,构建包含12×UAS重复基序的转基因质粒和辅助质粒,通过电泳迁移实验分析重组蛋白P2C-Gal4 DNA BINDING和12×UAS重复基序间的体外结合;分别将体外孵育的P2C-Gal4 DNA BINDING+辅助质粒ITF36-12×UAS和P2C-Gal4 DNA BINDING+转基因质粒ITF2-12×UAS afm复合物注射入吸血后20 h时的中华按蚊雌成蚊腹部,于血餐后40 h时提取其卵巢组织DNA,并通过特异性引物PCR扩增和测序分析外源DNA在活体中的投递情况。【结果】100%注射P2C-Gal4-DsRed的中华按蚊雌成蚊卵巢在绿色滤光片下呈现明显的红色荧光,表明P2C-Gal4-DsRed重组蛋白能够被高效地导入雌成蚊卵巢中;P2C-Gal4 DNA BINDING重组蛋白能够与12×UAS重复基序以及含有该重复基序片段的质粒稳定结合;分别有91%和93%的注射了P2C-Gal4 DNA BINDING+ITF36-12×UAS和P2C-Gal4 DNA BINDING+ITF2-12×UAS afm的雌成蚊卵巢组织中能够检测到外源DNA片段。【结论】在中华按蚊中成功建立了基于P2C卵巢导向肽和Gal4-12×UAS重复基序结合特性的外源DNA投递技术体系;通过此技术平台能够便捷、快速和高效地实现质粒等DNA分子在中华按蚊卵巢中的投递,这为进一步简化转基因、过表达及基因敲入等遗传操作奠定了基础。

【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.