Efficient CELI endonuclease production in Nicotiana benthamiana through transient expression and applications in detections of mutation and gene editing events

doi:10.1016/j.plantsci.2020.110469

CORC > 植物研究所 > 中国科学院植物研究所 > 中科院植物分子生理学重点实验室

	Efficient CELI endonuclease production in Nicotiana benthamiana through transient expression and applications in detections of mutation and gene editing events
	Yao, Xue-Feng ; Wu, Shengyang ; Guo, Lei 4; Liu, Chun-Ming
刊名	PLANT SCIENCE
	2020
卷号	296
关键词	Production of CELI endonuclease Optimization of CELI digestion Transient expression Detections of mismatch and gene editing
ISSN号	0168-9452
DOI	10.1016/j.plantsci.2020.110469
文献子类	Article
英文摘要	Rapid and low-cost methods of detecting mutations and polymorphisms are crucial for genotyping applications including mutagenesis and gene editing. S1 family endonucleases such as T7E1, EndoV and CELI can potentially be used in enzymatic mismatch detection. Among them, CELI has been shown to be effective in detecting mutations in Targeting Induced Local Lesions IN Genomes (TILLING). However, current method of CELI purification from celery is laborious, and challenging for many non-biochemical laboratories, and the presence of post-translational modifications hinders efficient production of the enzyme in E. coli. Here, we report an efficient system for bulk production of enzymatically active CELI endonuclease through transient expression in a model plant Nicotiana benthamiana. We also optimized the reaction buffer, by additions of Mn2+ and DTT, with enhanced mismatch cleavage activity. Using the new CELI production and reaction system, we were able to routinely detect mismatches in 1/32 mixed mutant and wildtype DNA samples. We believe the newly established system has many applications in characterization of mutations occurred in natural variations, mutagenized populations and gene editing.
学科主题	Biochemistry & Molecular Biology ; Plant Sciences
出版地	CLARE
WOS关键词	PLANTS ; CRISPR/CAS9 ; GENERATION ; PROTOCOL ; SYSTEM
WOS研究方向	Biochemistry & Molecular Biology ; Plant Sciences
语种	英语
出版者	ELSEVIER IRELAND LTD
WOS记录号	WOS:000541125700004
资助机构	National Transgenic Research Projects of China [2019ZX08010003-002-009, 2016ZX08009-001-006-002, 2016ZX08009-003-005]
内容类型	期刊论文
源URL	[http://ir.ibcas.ac.cn/handle/2S10CLM1/21715]
专题	中科院植物分子生理学重点实验室
作者单位	1.Chinese Acad Sci, Inst Bot, Key Lab Plant Mol Physiol, Beijing 100093, Peoples R China 2.Univ Chinese Acad Sci, Beijing 100049, Peoples R China 3.Wu, Shengyang 4.[Yao, Xue-Feng 5.Chinese Acad Agr Sci, Inst Crop Sci, Beijing 100081, Peoples R China
推荐引用方式 GB/T 7714	Yao, Xue-Feng,Wu, Shengyang,Guo, Lei,et al. Efficient CELI endonuclease production in Nicotiana benthamiana through transient expression and applications in detections of mutation and gene editing events[J]. PLANT SCIENCE,2020,296.
APA	Yao, Xue-Feng,Wu, Shengyang,Guo, Lei,&Liu, Chun-Ming.(2020).Efficient CELI endonuclease production in Nicotiana benthamiana through transient expression and applications in detections of mutation and gene editing events.PLANT SCIENCE,296.
MLA	Yao, Xue-Feng,et al."Efficient CELI endonuclease production in Nicotiana benthamiana through transient expression and applications in detections of mutation and gene editing events".PLANT SCIENCE 296(2020).