The invasive fly Drosophila suzukii has become an established fruit pest in Europe, the USA, and South America with no effective and safe pest management. Genetic engineering enables the development of transgene-based novel genetic control strategies against insect pests and disease vectors. This, however, requires the establishment of reliable germline transformation techniques. Previous studies have shown that D. suzukii is amenable to transgenesis using the transposon-based vectors piggyBac and Minos, site-specific recombination (lox/Cre), and CRISPR/Cas9 genome editing. We experienced differences in the usability of piggyBac-based germline transformation in different strains of D. suzukii: we obtained no transgenic lines in a US strain, a single rare transgenic line in an Italian strain, but observed a reliable transformation rate of 2.5 to 11% in a strain from the French Alps. This difference in efficiency was confirmed by comparative examination of these three strains. In addition, we used an attP landing site line to successfully established φC31-integrase-mediated plasmid integration at a rate of 10% and generated landing site lines with two attP sequences to effectively perform φC31-Recombinase Mediated Cassette Exchange (φC31-RMCE) with 11% efficiency. Moreover, we isolated and used the endogenous regulatory regions of Ds nanos to express φC31 integrase maternally to generate self-docking lines for φC31-RMCE. Besides, we isolated the promoter/enhancer of Ds serendipity α to drive the heterologous tetracycline-controlled transactivator (tTA) during early embryonic development and generated a testes-specific tTA driver line using the endogenous beta-2-tubulin (β2t) promoter/enhancer. Our results provide evidence that the D. suzukii strain AM derived from the French Alps is more suitable for piggyBac germline transformation than other strains. We demonstrated the feasibility of using φC31-RMCE in the cherry vinegar fly and generated a set of lines that can be used for highly efficient integration of larger constructs. The φC31-based integration will facilitate modification and stabilization of previously generated transgenic lines that carry at least one attP site in the transgene construction. An early embryo-specific and a spermatogenesis-specific driver line were generated for future use of the binary expression system tet-off to engineer tissue- and stage-specific effector gene expression for genetic pest control strategies.

中文翻译：

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入侵农业害虫，樱桃醋蝇，果蝇铃木的基因工程改进

入侵蝇果蝇铃木已成为欧洲，美国和南美的一种公认的果害，没有有效，安全的害虫管理方法。基因工程使人们能够开发基于转基因的新型遗传控制策略来对抗害虫和病媒。但是，这需要建立可靠的种系转化技术。先前的研究表明，使用基于转座子的载体piggyBac和Minos，位点特异性重组（lox / Cre）和CRISPR / Cas9基因组编辑，铃木D.适合转基因。我们在不同的铃木D.菌株中遇到了基于piggyBac的种系转化的可用性差异：我们在美国菌株中未获得转基因品系，在意大利菌株中仅获得了一种罕见的转基因品系，但观察到的可靠转化率为2。来自法国阿尔卑斯山的菌株的5至11％。通过对这三种菌株的比较检查，证实了效率的差异。此外，我们使用attP着陆位点序列以10％的比率成功建立了φC31整合酶介导的质粒整合，并生成具有两个attP序列的着陆点序列，以有效地进行φC31-重组酶介导的盒式交换（φC31-RMCE），效率11％。此外，我们分离并使用Ds nanos的内源性调控区来表达母体整合的φC31，以生成φC31-RMCE的自对接系。除了，我们分离了Ds偶然性α的启动子/增强子，以在早期胚胎发育过程中驱动异源四环素控制的反式激活因子（tTA），并使用内源性β-2-微管蛋白（β2t）启动子/增强子生成了睾丸特异性tTA驱动子系。我们的结果提供了证据，证明源自法国阿尔卑斯山的铃木D.铃木AM菌株比其他菌株更适合ggyBac种系转化。我们证明了在樱桃醋蝇中使用φC31-RMCE的可行性，并生成了一套可用于高效整合大型构建体的品系。基于φC31的整合将促进对先前产生的转基因品系的修饰和稳定化，这些转基因品系在转基因构建中带有至少一个atP位点。