Xu Na, Li Li, Chen Fusheng
Hubei International Scientific and Technological Cooperation Base of Traditional Fermented Foods, Huazhong Agricultural University, Wuhan, China.
College of Food Science and Technology, Huazhong Agricultural University, Wuhan, China.
Front Microbiol. 2022 Aug 1;13:952323. doi: 10.3389/fmicb.2022.952323. eCollection 2022.
spp. are traditional medicinal and edible filamentous fungi in China, and can produce various secondary metabolites, such as pigments (MPs) and citrinin (CIT). Genetic modification methods, such as gene knock-out, complementation, and overexpression, have been used extensively to investigate the function of related genes in spp.. However, the resistance selection genes that can have been used for genetic modification in spp. are limited, and the gene replacement frequency (GRF) is usually <5%. Therefore, we are committed to construct a highly efficient gene editing system without resistance selection marker gene. In this study, using M7 as the starting strain, we successfully constructed a so-called markerlessly and highly genetic modification system including the mutants ΔΔ and ΔΔ::, in which we used the endogenous gene from M7 instead of the resistance marker gene as the screening marker, and simultaneously deleted related to non-homologous end joining in M7. Then, the morphology, the growth rate, the production of MPs and CIT of the mutants were analyzed. And the results show that the mutant strains have normal mycelia, cleistothecia and conidia on PDA+Uridine(U) plate, the biomass of each mutant is also no different from M7. However, the U addition also has a certain effect on the orange and red pigments yield of M7, which needs our further study. Finally, we applied the system to delete multiple genes from M7 separately or continuously without any resistance marker gene, and found that the average GRF of ΔΔ was about 18 times of that of M7. The markerlessly and highly genetic modification system constructed in current study not only will be used for multi-gene simultaneous modification in spp., and also lays a foundation for investigating the effects of multi-genes modification on spp..
在中国，某些物种是传统的药用和食用丝状真菌，能够产生多种次级代谢产物，如色素（MPs）和桔霉素（CIT）。基因敲除、互补和过表达等基因改造方法已被广泛用于研究某些物种中相关基因的功能。然而，可用于某些物种基因改造的抗性选择基因有限，基因替换频率（GRF）通常<5%。因此，我们致力于构建一个无抗性选择标记基因的高效基因编辑系统。在本研究中，以M7作为出发菌株，我们成功构建了一个所谓的无标记且高效的基因改造系统，包括突变体ΔΔ和ΔΔ::，其中我们使用来自M7的内源基因代替抗性标记基因作为筛选标记，并同时删除了M7中与非同源末端连接相关的基因。然后，分析了突变体的形态、生长速率、MPs和CIT的产生情况。结果表明，突变菌株在PDA+尿苷（U）平板上具有正常的菌丝体、闭囊壳和分生孢子，每个突变体的生物量也与M7无差异。然而，添加U对M7的橙色和红色色素产量也有一定影响，这需要我们进一步研究。最后，我们应用该系统在无任何抗性标记基因的情况下分别或连续从M7中删除多个基因，发现ΔΔ的平均GRF约为M7的18倍。本研究构建的无标记且高效的基因改造系统不仅将用于某些物种的多基因同时改造，也为研究多基因改造对某些物种的影响奠定了基础。
