化学试剂对塔宾曲霉同源重组效率的影响

doi:10.19586/j.2095-2341.2024.0046

生物技术进展 ›› 2024, Vol. 14 ›› Issue (4): 586-593.DOI: 10.19586/j.2095-2341.2024.0046

• 研究论文 • 上一篇

化学试剂对塔宾曲霉同源重组效率的影响

梁丽存¹(), 刘文龙², 刘晓青¹, 姚斌¹, 黄火清¹, 杨浩萌¹()

^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

收稿日期:2024-03-07 接受日期:2024-04-18 出版日期:2024-07-25 发布日期:2024-08-07
通讯作者: 杨浩萌
作者简介:梁丽存E-mail： lianglicun1022@163.com；
基金资助:
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)

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

Licun LIANG¹(), Wenlong LIU², Xiaoqing LIU¹, Bin YAO¹, Huoqing HUANG¹, Haomeng YANG¹()

^1.Institute of Animal Sciences，Chinese Academy of Agricultural Sciences，Beijing 100193，China
^2.Longkete Enzyme Preparations，Shandong Linyi 276000，China

Received:2024-03-07 Accepted:2024-04-18 Online:2024-07-25 Published:2024-08-07
Contact: Haomeng YANG

摘要/Abstract

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。

关键词: 化学试剂, 同源重组效率, 塔宾曲霉

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 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.

Key words: chemical reagents, homologous recombination efficiency, Aspergillus tubingensis

中图分类号:

Q784

梁丽存, 刘文龙, 刘晓青, 姚斌, 黄火清, 杨浩萌. 化学试剂对塔宾曲霉同源重组效率的影响[J]. 生物技术进展, 2024, 14(4): 586-593.

Licun LIANG, Wenlong LIU, Xiaoqing LIU, Bin YAO, Huoqing HUANG, Haomeng YANG. The Effect of Chemical Reagents on the Efficiency of Gene Editing in Aspergillus tubingensis[J]. Current Biotechnology, 2024, 14(4): 586-593.

图/表 7

参考文献 20

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小分子名称	靶点	工作原理	作用效果	参考文献
RS-1	RAD51	激活RAD51，稳定ssDNA单链	增强CRISPR/Cas介导的敲入效率	[14]
SCR7	DNA LigaseⅣ	抑制DNA LigaseⅣ 活性	抑制NHEJ途径	[15]
NU7441(KU-57788)	DNA-PKcs	降低DNA-PKcs蛋白的表达	抑制NHEJ途径	[16]
Vanillin	DNA-PKcs	抑制DNA-PKcs蛋白的活性	抑制NHEJ途径	[17]
Chlorpromazine	KU70/80	抑制KU蛋白辅因子的产生	抑制NHEJ途径	[17]
Resveratrol	DNA LigaseⅣ	抑制DNA LigaseⅣ活性	抑制NHEJ途径	[18]
Benomyl	细胞有丝分裂	阻滞细胞分裂M期	细胞周期同步化	[13]
Inositol	细胞壁的形成	促进真菌细胞壁的形成	增加单核原生质体的形成	[13]

小分子名称	靶点	工作原理	作用效果	参考文献
RS-1	RAD51	激活RAD51，稳定ssDNA单链	增强CRISPR/Cas介导的敲入效率	[14]
SCR7	DNA LigaseⅣ	抑制DNA LigaseⅣ 活性	抑制NHEJ途径	[15]
NU7441(KU-57788)	DNA-PKcs	降低DNA-PKcs蛋白的表达	抑制NHEJ途径	[16]
Vanillin	DNA-PKcs	抑制DNA-PKcs蛋白的活性	抑制NHEJ途径	[17]
Chlorpromazine	KU70/80	抑制KU蛋白辅因子的产生	抑制NHEJ途径	[17]
Resveratrol	DNA LigaseⅣ	抑制DNA LigaseⅣ活性	抑制NHEJ途径	[18]
Benomyl	细胞有丝分裂	阻滞细胞分裂M期	细胞周期同步化	[13]
Inositol	细胞壁的形成	促进真菌细胞壁的形成	增加单核原生质体的形成	[13]

引物名称	引物序列（5′→3′）	用途
fwn-up-500-F	5′-CAACCAAGCTCAAGGTTCCTTACGCG-3′	扩增500 bp同源臂的供体DNA
fwn-dw-500-R	5′-TCCGCAGCAGCGCAGTCAAAG-3′	扩增500 bp同源臂的供体DNA
fwn-dw-F	5′-CTATACCAACAACTTCTGCCTGAGCAAGG-3′	扩增上下游同源臂
fwn-up-R	5′-GTTCTTGAGATCCCACTTGTAGGCTGGG-3′	扩增上下游同源臂
hph-fu-F	5′-CAGGCTACAAGTGGGATCTCAAGAACGACAGAAGATGATATTGAAGGAGCACTTTTTG-3′	融合潮霉素基因
hph-fu-R	5′-AGTTGTTGGTATAGGGAATCCAGTAGTATCTGGAAGAGGTAAACCCGAAACG-3′	融合潮霉素基因
500-fwn-F	5′-CGATGCTGGCCATCAAGGCGTCCCTGGC-3′	检测转化子
HPH-150-R	5′-GCTACTGCTACAAGTGGGGCTGATCTGACC-3′	检测转化子
5SRNA-F	5′-CACATACGACCACAGGGTGTG-3′	扩增gRNA表达盒
gRNA-R	5′-AAAAGCACCGACTCGGTGCC-3′	扩增gRNA表达盒
Cas9-F	5′-CGAGACAGCAGAATCACCGCCCAAGTTAAG-3′	扩增Cas9表达盒
Cas9-R	5′-ATTACACTTGTATTGGGATGAATTTTGTATGCAC-3′	扩增Cas9表达盒

引物名称	引物序列（5′→3′）	用途
fwn-up-500-F	5′-CAACCAAGCTCAAGGTTCCTTACGCG-3′	扩增500 bp同源臂的供体DNA
fwn-dw-500-R	5′-TCCGCAGCAGCGCAGTCAAAG-3′	扩增500 bp同源臂的供体DNA
fwn-dw-F	5′-CTATACCAACAACTTCTGCCTGAGCAAGG-3′	扩增上下游同源臂
fwn-up-R	5′-GTTCTTGAGATCCCACTTGTAGGCTGGG-3′	扩增上下游同源臂
hph-fu-F	5′-CAGGCTACAAGTGGGATCTCAAGAACGACAGAAGATGATATTGAAGGAGCACTTTTTG-3′	融合潮霉素基因
hph-fu-R	5′-AGTTGTTGGTATAGGGAATCCAGTAGTATCTGGAAGAGGTAAACCCGAAACG-3′	融合潮霉素基因
500-fwn-F	5′-CGATGCTGGCCATCAAGGCGTCCCTGGC-3′	检测转化子
HPH-150-R	5′-GCTACTGCTACAAGTGGGGCTGATCTGACC-3′	检测转化子
5SRNA-F	5′-CACATACGACCACAGGGTGTG-3′	扩增gRNA表达盒
gRNA-R	5′-AAAAGCACCGACTCGGTGCC-3′	扩增gRNA表达盒
Cas9-F	5′-CGAGACAGCAGAATCACCGCCCAAGTTAAG-3′	扩增Cas9表达盒
Cas9-R	5′-ATTACACTTGTATTGGGATGAATTTTGTATGCAC-3′	扩增Cas9表达盒

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

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