mitoTALENs植物线粒体基因编辑载体的构建方法

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

摘要/Abstract

摘要：

【目的】mitoTALENs植物线粒体基因编辑技术能够高效地实现线粒体基因的敲除，进而有效实现线粒体基因功能的研究，但mitoTALENs载体构建过程非常的繁琐复杂且目前仍没有较为系统完整的载体构建方法作为参考。为解决这个问题，结合前人已发表的及本实验室摸索出的方法对mitoTALENs载体构建的完整过程进行了详尽描述，为之后利用mitoTALENs技术进行植物线粒体基因功能研究的研究者们提供重要参考。【方法】以水稻线粒体WA352基因作为目的基因，利用其序列特异性区域设计了靶点TAL，首先采用Platinum gate TALEN assembly的两步组装技术分别构建了mitoTALENs的TALEN-left和TALEN-right载体，然后利用multisite LR反应将TALEN-left、TALEN-right及含有其他功能元件的进入载体和目的载体进行反应，生成最终的表达载体。【结果】第一步组装构建10个载体，第二步组装构建2个载体，最后通过multisite LR反应构建1个终表达载体。【结论】详细介绍了mitoTALENs载体的构建过程，为该技术使用者提供重要参考，以促进植物线粒体基因编辑研究领域的发展。

关键词: 线粒体基因编辑, mitoTALENs载体, Platinum gate TALEN assembly, multisite LR

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

周家伟, 武志强. mitoTALENs植物线粒体基因编辑载体的构建方法[J]. 生物技术通报, 2024, 40(10): 172-180.

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

图/表 14

图1 TALENs的结构加粗的HD及方框中的NI、HD、NG、NN和NK均表示重复可变双残基（RVD）

Fig. 1 Structure of TALENs The bold HD and the NI, HD, NG, NN, and NK in the box all indicate repeat variable diresidues（RVD）

图2 mitoTALENs技术的工作示意图

Fig. 2 Schematic diagram of mitoTALENs technology

图3 mitoTALENs载体构建原理图 A：TALE阵列的两步组装原理图；B：multisite LR反应原理图

Fig. 3 Schematic diagram of mitoTALENs vector construction A: Two-step assembly schematic diagram of TALE array. B: Schematic diagram multisite LR reaction

表1 mitoTALENs载体构建所需的质粒载体

Table 1 Plasmid vectors required for the construction of mitoTALENs vector

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

表2 第一步组装的反应体系

Table 2 Reaction system in the first step assembly

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

图4 第一步组装方法示意图 A：WA352基因的靶点；B：TALE阵列的第一步组装方法

Fig. 4 Schematic diagram of the first step assembly method A: The target of WA352 gene. B: The first step assembly method for TALE arrays

表3 第二步组装的反应体系

Table 3 Reaction system in the second step assembly

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

表4 引物列表

Table 4 Primers list

引物名称Primer name	引物序列Primer sequence（5'-3'）	目的Purpose
Step1_F	TTGATGCCTGGCAGTTCCCT	第一步组装结果检测Step 1 assembly result detection
Step1_R	CGAACCGAACAGGCTTATGT
Step2_F	GGACCGTCGCTGTCAAGTATCA	第二步组装结果检测 Step 2 assembly result detection
Step2_R	AAGAACTCCATCACCTTCATCTCCAG
attB1_F	ACAAGTTTGTACAAAAAAGCAGGCT	终载体组装结果检测 Result detection of final vectorr assembly
attB4_R	CAACTTTGTATAGAAAAGTTGGGTGTCT
attB4_F	AGACACCCAACTTTTCTATACAAAGTTG
attB3_R	CAACTTTATTATACAAAGTTGTGGAATCGAGC
attB3_F	GCTCGATTCCACAACTTTGTATAATAAAGTTG
attB2_R	ACCACTTTGTACAAGAAAGCTGG

表5 本研究构建的载体

Table 5 Vectors constructed in this study

目的基因 Target gene	靶点 Target	第一步组装 Step 1	第二步组装 Step 2	多位点LR Multisite LR
WA352	TAL	pFUS2_a4a（L1）, pFUS2_a4b（L2）, pFUS2_a3b（L3）, pFUS2_a2b（L4）, pFUS2_b3（L5）	pENTR E1 pF5A L1-L4（last HD）	WA352::mitoTALEN
		pFUS2_a4a（R1）, pFUS2_a4b（R2）, pFUS2_a3b（R3）, pFUS2_a2b（R4）, pFUS2_b3（R5）	pENTR E3 pR5A L3-L2（last HD）	WA352::mitoTALEN
总数Total		10	2	1

图5 第一步组装结果的阳性检测 A：蓝白斑筛选阳性单克隆；B：L1-L5和R1-R5这10个载体的PCR阳性检测

Fig. 5 Positive detection of assembly results in the first step A: Screening positive monoclonal via blue and white spot. B: The ten vectors L1-L5 and R1-R5 were detected by PCR

图6 第二步组装结果的PCR检测 WA352::TALEN-left（L）和WA352::TALEN-right（R）的PCR检测

Fig. 6 PCR detection of assembly results in the second step PCR detection of WA352::TALEN-left（L）and WA352::TALEN-right（R）

图7 第二步组装结果的酶切检测 A：WA352基因的TAL-left（L）和TAL-right（R）所对应的重复可变双残基（RVDs），其中NI是Msc I的消化位点；B：TAL-left（L）和TAL-right（R）经Msc I消化后的产物胶图

Fig. 7 Enzyme digestion of the assembly results in the second step A: The repeat variable diresidues（RVDs）corresponding to TAL-left（L）and TAL-right（R）of WA352 gene, where NI is the digestion site of Msc I. B: Gel image of the products of TAL-left（L）and TAL-right（R）digested by Msc I

图8 终表达载体的PCR检测

Fig. 8 PCR detection of the final expression vector

图9 WA352::mitoTALEN载体图

Fig. 9 Diagram of WA352::mitoTALEN vector

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