Construction Method of mitoTALENs Mitochondrial Gene Editing Vector in Plants

doi:10.13560/j.cnki.biotech.bull.1985.2024-0657

Abstract

Abstract:

【Objective】 mitoTALENs plant mitochondrial gene editing technology can effectively achieve the knockout of mitochondrial genes, and then effectively conduct the study of mitochondrial gene function. However, the vector construction process of mitoTALENs is very complicated and there is still no relatively systematic and complete vector construction method as a reference. To solve this issue, we described in detail the complete process of mitoTALENs vector construction in combination with the methods published by predecessors and explored by ourselves, providing an important reference for researchers who use mitoTALENs technology to study plant mitochondrial gene function. 【Method】 With rice mitochondrial WA352 gene as the target gene, a target TAL was designed using its sequence-specific region. Firstly, the TALEN-left and TALEN-right vectors of mitoTALENs were constructed using the two-step assembly technology of Platinum gate TALEN assembly, respectively. Then, multisite LR reaction was used to react TALEN-left, TALEN-right, the entry vector containing other functional elements, and the destination vector to generate the final expression vector.【Result】 Ten vectors were assembled in the first step, two vectors were assembled in the second step, and one final expression vector was constructed by multisite LR reaction.【Conclusion】 The detailed introduction of mitoTALENs vector construction process provides an important reference for users and promotes the development of plant mitochondrial gene editing research.

Key words: mitochondrial gene editing, mitoTALENs vector, Platinum gate TALEN assembly, multisite LR

ZHOU Jia-wei, WU Zhi-qiang. Construction Method of mitoTALENs Mitochondrial Gene Editing Vector in Plants[J]. Biotechnology Bulletin, 2024, 40(10): 172-180.

Figures/Tables 14

References 18

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载体类型 Vector type	载体内容 Vector content	载体原核抗性 Vector prokaryotic resistance
模块质粒Module plasmids	p1NI，p2NI，p3NI，p4NI，p1NG，p2NG，p3NG，p4NG，p1HD，p2HD，p3HD，p4HD，p1NN，p2NN，p3NN，p4NN	AmpR
中间阵列质粒 Intermediate array plasmids	pFUS2_a1a，pFUS2_a2a，pFUS2_a2b，pFUS2_a3a，pFUS2_a3b，pFUS2_a4a，pFUS2_a4b，pFUS2_b1，pFUS2_b2，pFUS2_b3，pFUS2_b4	SpeR
进入载体1 Entry vectors 1	pENTR E1 pF5A L1-L4，pENTR E1 pF6A L1-L4， pENTRE1 pF7A L1-L4，pENTR E1 pF8A L1-L4	KanR
进入载体2 Entry vectors 2	pENTER E2 HSPter-35Sp-mito	KanR
进入载体3 Entry vectors 3	pENTR E3 pR5A L3-L2，pENTR E3 pR6A L3-L2， pENTR E3 pR7A L3-L2，pENTR E3 pR8A L3-L2	KanR
目的载体 Destination vector	pDESTpK7WG2mito	SpeR

载体类型 Vector type	载体内容 Vector content	载体原核抗性 Vector prokaryotic resistance
模块质粒Module plasmids	p1NI，p2NI，p3NI，p4NI，p1NG，p2NG，p3NG，p4NG，p1HD，p2HD，p3HD，p4HD，p1NN，p2NN，p3NN，p4NN	AmpR
中间阵列质粒 Intermediate array plasmids	pFUS2_a1a，pFUS2_a2a，pFUS2_a2b，pFUS2_a3a，pFUS2_a3b，pFUS2_a4a，pFUS2_a4b，pFUS2_b1，pFUS2_b2，pFUS2_b3，pFUS2_b4	SpeR
进入载体1 Entry vectors 1	pENTR E1 pF5A L1-L4，pENTR E1 pF6A L1-L4， pENTRE1 pF7A L1-L4，pENTR E1 pF8A L1-L4	KanR
进入载体2 Entry vectors 2	pENTER E2 HSPter-35Sp-mito	KanR
进入载体3 Entry vectors 3	pENTR E3 pR5A L3-L2，pENTR E3 pR6A L3-L2， pENTR E3 pR7A L3-L2，pENTR E3 pR8A L3-L2	KanR
目的载体 Destination vector	pDESTpK7WG2mito	SpeR

模块质粒的个数 Number of module plasmids	中间阵列质粒 Intermediate array plasmids	模块质粒 Module plasmids	T4 DNA ligase reaction buffer	Bsa I-HF	Quick ligase	ddH₂O	总体积 Total volume/µL
1	0.3	0.3×1	0.2	0.1	0.1	1	2
2	0.3	0.3×2	0.2	0.1	0.1	0.7	2
3	0.3	0.3×3	0.2	0.1	0.1	0.4	2
4	0.3	0.3×4	0.2	0.1	0.1	0.1	2

模块质粒的个数 Number of module plasmids	中间阵列质粒 Intermediate array plasmids	模块质粒 Module plasmids	T4 DNA ligase reaction buffer	Bsa I-HF	Quick ligase	ddH₂O	总体积 Total volume/µL
1	0.3	0.3×1	0.2	0.1	0.1	1	2
2	0.3	0.3×2	0.2	0.1	0.1	0.7	2
3	0.3	0.3×3	0.2	0.1	0.1	0.4	2
4	0.3	0.3×4	0.2	0.1	0.1	0.1	2

模块质粒的个数 Number of module plasmids	pFUS2_a质粒 pFUS2_a plasmids	pFUS2_b质粒 pFUS2_b plasmids	入门载体 Entry vectors	T4 DNA ligase reaction buffer	Esp3 I	Quick ligase	ddH₂O	总体积 Total volume/µL
6-9	0.6×1	0.6	0.3	0.4	0.2	0.2	1.7	4
10-13	0.6×2	0.6	0.3	0.4	0.2	0.2	1.1	4
14-17	0.6×3	0.6	0.3	0.4	0.2	0.2	0.5	4
18-21	0.6×4	0.6	0.3	0.4	0.2	0.2	0	4.1