Dong Zhanqi, Gao Na, Deng Boyuan, Huang Xuhua, Hu Congwu, Chen Peng, Wu Qin, Lu Cheng, Pan Minhui
State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, 400716, China.
Key Laboratory of Sericultural Biology and Genetic Breeding, Ministry of Agriculture, Southwest University, Chongqing, 400716, China.
Parasit Vectors. 2022 Apr 21;15(1):141. doi: 10.1186/s13071-022-05236-4.
Microsporidia are a group of intracellular parasitic eukaryotes, serious pathogens that cause widespread infection in humans, vertebrates, and invertebrates. Because microsporidia have a thick spore wall structure, the in vitro transformation, cell culture, and genetic operation technology of microsporidia are far behind that of other parasites.
In this study, according to an analysis of the life-cycle of microsporidia, Nosema bombycis, and different electro-transformation conditions, the transduction efficiency of introducing foreign genes into N. bombycis was systematically determined.
We analyzed the direct electro-transformation of foreign genes into germinating N. bombycis using reporters under the regulation of different characteristic promoters. Furthermore, we systematically determined the efficiency of electro-transformation into N. bombycis under different electro-transformation conditions and different developmental stages through an analysis of the whole life-cycle of N. bombycis. These results revealed that foreign genes could be effectively introduced through a perforation voltage of 100 V pulsed for 15 ms during the period of N. bombycis sporeplasm proliferation.
We present an effective method for electro-transformation of a plasmid encoding a fluorescent protein into N. bombycis, which provides new insight for establishing genetic modifications and potential applications in these intracellular parasites.
微孢子虫是一类细胞内寄生的真核生物，是导致人类、脊椎动物和无脊椎动物广泛感染的严重病原体。由于微孢子虫具有厚的孢子壁结构，其体外转化、细胞培养和基因操作技术远远落后于其他寄生虫。
在本研究中，根据对微孢子虫、家蚕微孢子虫的生命周期及不同电转化条件的分析，系统地测定了将外源基因导入家蚕微孢子虫的转导效率。
我们使用在不同特征启动子调控下的报告基因，分析了将外源基因直接电转化到家蚕微孢子虫萌发孢子中的情况。此外，通过对家蚕微孢子虫整个生命周期的分析，我们系统地测定了在不同电转化条件和不同发育阶段将外源基因电转化到家蚕微孢子虫中的效率。这些结果表明，在家蚕微孢子虫孢子质增殖期间，通过100V脉冲15ms的穿孔电压可有效导入外源基因。
我们提出了一种将编码荧光蛋白的质粒电转化到家蚕微孢子虫中的有效方法，这为在这些细胞内寄生虫中建立基因修饰和潜在应用提供了新的见解。
