植物线粒体基因组编辑研究进展

doi:10.13560/j.cnki.biotech.bull.1985.2024-0638

摘要/Abstract

摘要：

线粒体是真核生物细胞内的半自主细胞器，具有自身的基因组（mtDNA），在生命活动中扮演重要角色。人类mtDNA突变与多种遗传疾病相关，而在植物中mtDNA高频重组产生的ORF基因常常与植株雄性不育表型相关。随着基因编辑技术的迅速发展，mtDNA编辑技术为研究和治疗线粒体疾病提供了有力工具。得益于mtDNA编辑工具的丰富，线粒体编辑技术也被广泛应用于植物线粒体基因组功能基因以及未知序列的研究中。相较于核基因组编辑，针对mtDNA编辑仍然面临一些限制因素。本文总结了mtDNA编辑技术的发展和研究现状，以及在植物领域利用这些编辑技术对mtDNA进行的研究进展，展望了在植物线粒体研究中的mtDNA编辑技术潜在优化思路以及应用潜力。

关键词: 线粒体, 基因编辑, ZFN, TALEN, CRISPR

Abstract:

Mitochondria, the semi-autonomous organelles within eukaryotic cells, harbor specific genomes（mtDNA）and play pivotal roles in cellular life processes. mtDNA mutations in human are linked to a spectrum of genetic disorders, while in plants, recombination in mtDNA often leads to ORF genes associated with male sterility. The advent of gene editing technologies has revolutionized the study and treatment of mitochondrial diseases. With a plethora of mtDNA editing tools available, these technologies have also been instrumental in exploring functional genes and unknown sequences within plant mitochondrial genomes. Compared to nuclear genome editing, mtDNA editing still encounters certain challenges. This paper provides an overview of the development and current state of mtDNA editing technologies, examines their application in plant research, and contemplates future optimization strategies and the potential impact of these technologies on plant mitochondrial studies.

Key words: mitochondria, gene editing, ZFN, TALEN, CRISPR

张硕, 阚俊虎, 周家伟, 武志强. 植物线粒体基因组编辑研究进展[J]. 生物技术通报, 2024, 40(10): 41-52.

ZHANG Shuo, KAN Jun-hu, ZHOU Jia-wei, WU Zhi-qiang. Advance in Plant Mitochondrial Genome Editing[J]. Biotechnology Bulletin, 2024, 40(10): 41-52.

图/表 3

图1 线粒体基因组编辑技术示意图 a: mitoZFN中串联的ZFP锌指蛋白特异识别靶序列，Fok I形成的二聚体具有核酸酶活性；b: mitoTALEN中串联的TALE蛋白特异识别靶序列，Fok I形成的二聚体具有核酸酶活性；c：mito-Cas9中Cas9蛋白可以借助MTS进入线粒体内，而sgRNA和单链寡核苷酸只能有限的进入线粒体内，限制了mito-Cas9的编辑效率

Fig. 1 Schematic diagram of mitochondrial genome editing technology a: In mitoZFN, tandem ZFP zinc finger proteins specifically recognize target sequences, and the Fok I dimer exhibits nuclease activity. b: In mitoTALEN, tandem TALE proteins specifically recognize target sequences, and the Fok I dimer exhibits nuclease activity. c: In mito-Cas9, the Cas9 protein can enter the mitochondria with the help of MTS, while sgRNA and single-stranded oligonucleotides can only enter the mitochondria to a limited extent, restricting the editing efficiency of mito-Cas9

图2 在植物中应用的线粒体基因组编辑技术示意图 a: mitoTALEN经过密码子优后应用到植物线粒体研究中；b: mt-DdCBEs/mitoTALECD利用串联的TALE蛋白特异识别靶序列，DddA脱氨酶将胞嘧啶转化为尿嘧啶，UGI作为尿嘧啶糖基化酶抑制剂保护尿嘧啶，经过DNA复制实现C-to-T的编辑；c：TALED中利用DddA脱氨酶打开双链DNA，具有单链特异性TadA8e腺嘌呤脱氨酶将腺嘌呤转化为次黄嘌呤，经过DNA复制实现A-to-G的编辑；d：mitoCRISPR/Cas9中Cas9蛋白借助MTS进入线粒体内与sgRNA结合实现对靶序列的切割

Fig. 2 Schematic representation of mitochondrial genome editing techniques applied in plants a: Codon-optimized mitoTALEN has been applied to plant mitochondrial research. b: mt-DdCBEs/mitoTALECD utilizes tandem TALE proteins to specifically recognize target sequences. The DddA deaminase converts cytosine to uracil, and UGI acts as a uracil glycosylase inhibitor to protect uracil, achieving C-to-T editing through DNA replication. c: In TALED, DddA deaminase opens double-stranded DNA, and the single-strand-specific TadA8e adenine deaminase converts adenine to inosine, achieving A-to-G editing through DNA replication. d: In mitoCRISPR/Cas9, the Cas9 protein, aided by MTS, enters the mitochondria and binds with sgRNA to achieve target sequence cleavage

表1 线粒体基因组编辑技术在植物线粒体基因功能研究中的应用

Table 1 Application of mitochondrial genome editing technology in the study of plant mitochondrial gene functions

编辑工具 Editing tool	物种 Species	靶基因 Target gene	编辑类型 Editing type	表型 Phenotype	文献 Reference
mitoTALEN	水稻Oryza sativa	orf79	敲除	恢复花粉育性	[61]
mitoTALEN	油菜 Brassica napus	orf125	敲除	恢复花粉育性	[61]
mitoTALEN	拟南芥Arabidopsis thaliana	atp6-1, atp6-2	敲除	功能冗余；双突致死	[69]
mitoTALEN	水稻Oryza sativa	orf352	敲除	恢复花粉育性；不结实	[62]
mitoTALEN	水稻Oryza sativa	orf312	敲除	恢复花粉育性	[63]
mitoTALEN	番茄Solanum lycopersicum	orf137	敲除	恢复花粉育性	[65]
TALEN-GDM	烟草 Nicotiana tabacum	nad9	点突变	无明显表型	[71]
TALEN-GDM	烟草Nicotiana tabacum	nad9	敲除	生长迟缓；叶片和花发育缺陷；雄性不育	[72]
mitoTALEN	拟南芥Arabidopsis thaliana	nad7	敲除	严重的生长迟缓；致死	[70]
mitoTALEN	水稻Oryza sativa	WA352	敲除	恢复花粉育性	[64]
mitoTALEN	西兰花Brassica oleracea	orf138	敲除	恢复花粉育性	[66]
mitoTALEN	马铃薯Solanum tuberosum	orf125	敲除	N/A	[79]
mt-DdCBEs	油菜Brassica napus	atp6, rps14	C-to-T	N/A	[73]
mt-DdCBEs	莴苣Lactuca sativa	atp6	C-to-T	N/A	[73]
mitoTALECD	拟南芥Arabidopsis thaliana	atp1	C-to-T	恢复正常生长	[74]
mitoTALECD	马铃薯Solanum tuberosum	orf125	C-to-T	N/A	[79]
mTALED	水稻Oryza sativa	atp6-2	A-to-G	N/A	[77]
mitoCRISPR/Cas9	烟草Nicotiana tabacum	atp9	敲除	雄性不育	[78]

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