化学试剂对塔宾曲霉同源重组效率的影响

The Effect of Chemical Reagents on the Efficiency of Gene Editing in Aspergillus tubingensis

梁丽存 ¹刘文龙 ²刘晓青 ¹姚斌 ¹黄火清 ¹杨浩萌¹

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作者信息

1. 中国农业科学院北京畜牧兽医研究所,北京 100193
2. 山东隆科特酶制剂有限公司,山东临沂 276000
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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.

Abstract

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 pro-toplast 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.

关键词

化学试剂/同源重组效率/塔宾曲霉

Key words

chemical reagents/homologous recombination efficiency/Aspergillus tubingensis

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基金项目

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)

出版年

2024

生物技术进展

中国农业科学院茶叶研究所中国农业科学院生物科技研究所

生物技术进展

CSTPCD

影响因子：0.554

ISSN：2095-2341

参考文献量2

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