摘要
某些化学试剂可以通过调节细胞周期或胞内DNA修复途径来提高基因编辑效率。以塔宾曲霉作为研究对象,通过CRISPR/Cas9工具,对其孢子色素合成相关基因fwn进行基因编辑,在转化过程中分别加入8种化学试剂,最后通过统计孢子颜色和抗性基因PCR验证,检测8种化学试剂对塔宾曲霉同源重组效率的影响。结果发现,与零添加对照组相比,在原生质体转化过程中,添加苯菌灵(benomyl)、RS-1、香草醛(vanillin)和氯丙嗪(chlorpromazine),同源重组效率分别达到85.45%、60.90%、59.45%、65.95%,比对照组提高了18.05%、9.35%、7.9%、14.4%;当苯菌灵与其他试剂叠加使用时,同源重组效率进一步提高,最高可达97.92%。因此,化学试剂在原生质体转化中的应用为提高丝状真菌的基因编辑效率提供了新的思路。
Some chemical reagents can improve gene editing efficiency by regulating cell cycle or intracellular DNA repair pathways.Using Aspergillus tubingensis as the research material,the gene fwn related to spore pigment synthesis was edited by CRISPR/Cas9 tool.Eight chemical reagents were added during the transformation process.Finally,the effects of eight chemical reagents on the gene editing efficiency of Aspergillus tubingensis were detected by spore color statistics and resistance gene PCR verification.The results showed that,compared with the control group,adding benomyl,RS-1,vanillin and chlorpromazine in the process of protoplast transformation,the homologous recombination efficiency reached 85.45%,60.90%,59.45%and 65.95%,respectively,which was 18.05%,9.35%,7.9%and 14.4%higher than that of the control group.When benomyl was used in combination with other reagents,the homologous recombination efficiency was further improved,up to 97.92%.Therefore,the application of chemical reagents in protoplast transformation provides a new idea for improving the gene editing efficiency of filamentous fungi.
作者
梁丽存
刘文龙
刘晓青
姚斌
黄火清
杨浩萌
LIANG Licun;LIU Wenlong;LIU Xiaoqing;YAO Bin;HUANG Huoqing;YANG Haomeng(Institute of Animal Sciences,Chinese Academy of Agricultural Sciences,Beijing 100193,China;Longkete Enzyme Preparations,Shandong Linyi 276000,China)
出处
《生物技术进展》
2024年第4期586-593,共8页
Current Biotechnology
基金
国家重点研发计划项目(2021YFC2104805)
国家现代农业产业技术体系专项(CARS-41)。
关键词
化学试剂
同源重组效率
塔宾曲霉
chemical reagents
homologous recombination efficiency
Aspergillus tubingensis
