Aynalem Tessema, Meng Lifeng, Getachew Awraris, Wu Jiangli, Yu Huimin, Tan Jing, Li Nannan, Xu Shufa
Key Laboratory of Pollinating Insect Biology, Ministry of Chinese Agriculture and Rural Affairs, Institute of Apicultural Research, Chinese Academy of Agricultural Sciences, Beijing 100093, China.
College of Agriculture and Environmental Science, Bahir Dar University, Bahir Dar P.O. Box 26, Ethiopia.
Microorganisms. 2022 Oct 21;10(10):2088. doi: 10.3390/microorganisms10102088.
is the causative agent of honey bee chalkbrood disease, and spores are the only known source of infections. Interference with sporulation is therefore a promising way to manage The versicolorin reductase gene () is a ketoreductase protein related to sporulation and melanin biosynthesis. To study the gene in ascospore production of , CRISPR/Cas9 was used, and eight hygromycin B antibiotic-resistant transformants incorporating enhanced green fluorescent protein () were made and analyzed. PCR amplification, gel electrophoresis, and sequence analysis were used for target gene editing analysis and verification. The CRISPR/Cas9 editing successfully knocked out the gene in mutants had shown albino and non-functional spore-cyst development and lost effective sporulation. In conclusion, editing of gene has shown direct relation with sporulation and melanin biosynthesis of ; this effective sporulation reduction would reduce the spread and pathogenicity of to managed honey bee. To the best of our knowledge, this is the first time CRISPR/Cas9-mediated gene editing has been efficiently performed in , a fungal honey bee brood pathogen, which offers a comprehensive set of procedural references that contributes to gene function studies and consequent control of chalkbrood disease.
是蜜蜂白垩病的病原体，孢子是唯一已知的感染源。因此，干扰孢子形成是一种有前景的病害管理方法。杂色曲霉素还原酶基因（）是一种与孢子形成和黑色素生物合成相关的酮还原酶蛋白。为了研究该基因在子囊孢子产生中的作用，使用了CRISPR/Cas9技术，构建并分析了8个整合增强型绿色荧光蛋白（）的潮霉素B抗性转化体。采用聚合酶链反应（PCR）扩增、凝胶电泳和序列分析进行靶基因编辑分析和验证。CRISPR/Cas9编辑成功敲除了该基因，突变体表现出白化和无功能的孢子囊发育，失去了有效的孢子形成能力。总之，基因编辑与的孢子形成和黑色素生物合成直接相关；这种有效的孢子形成减少将降低其向养殖蜜蜂传播的可能性和致病性。据我们所知，这是首次在蜜蜂幼虫病原真菌中高效进行CRISPR/Cas9介导的基因编辑，为基因功能研究和随后的白垩病防治提供了一套全面的程序参考。
