Construction and Application of Potato Mitochondrial Targeted Expression Vector

doi:10.13560/j.cnki.biotech.bull.1985.2023-1178

Abstract

Abstract:

【Objective】The genetic variations of mitochondria genome usually lead to cytoplasmic male sterility（CMS）in plants. Biotechnology is an important measure to produce variations resulting in CMS lines. In order to establish an efficient expression vector system for the research on potato mitochondrial genes, the optimal potato mitochondrial targeting expression vector was explored by comparing the expression level of eGFP, which was promoted by different combinations of promoters and mitochondrial targeting signal peptides（MTS）. 【Method】Three promoters AtUBI10, StUBI10, 2×35S and two mitochondrial transport signal peptides ATPγ and Rf1b were tested. The expression level of the target vector was verified by tobacco leaf transient expression system and PEG-mediated potato protoplast transformation system. 【Result】AtUBI10 :: ATPγ-eGFP among 6 expressing vectors had the best expression effect in tobacco and was accurately located in mitochondria. The second best ones were StUBI10 :: ATPγ-eGFP and 2×35S :: ATPγ-eGFP. The vector AtUBI10 :: ATPγ-eGFP also showed the best performance, followed by StUBI10 :: Rf1b-eGFP and AtUBI10 :: Rf1b-eGFP in the potato protoplast transformation system. The two vectors with 2×35S promoter were weakly expressed in protoplasts. 【Conclusion】The comparative studies showed the mitochondrial vector constructed by the promoter AtUBI10 and the mitochondrial transport signal peptide ATPγ is the best combination, and this vectors will be of great benefit to the relative study on mitochondrial genes.

Key words: potato, mitochondrial transport signal peptides, targeted expression vector, subcellular localization, cytoplasmic male sterility

ZHANG Zhen, LI Qing, XU Jing, CHEN Kai-yuan, ZHANG Chun-zhi, ZHU Guang-tao. Construction and Application of Potato Mitochondrial Targeted Expression Vector[J]. Biotechnology Bulletin, 2024, 40(5): 66-73.

Figures/Tables 6

References 32

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扩增片段Amplified fragment		引物序列Primer sequence（5'-3'）
2×35S启动子组合	2×35S	F	TATGACCATGATTACGAATTCGCTAGAGCAGCTTGCCAACATG
	2×35S	R	CCATTGCCATGGATCCAAGCTTGAAGAGAGAGAC
	2×35S	R*	GGCGTGCCATGGTCGATCGACAGATCTGCG
	ATPγ	F	TCGATCGACCATGGCAATGGCTGTTTTCCG
	ATPγ	R	TGCTCACCATGCCTTGAACTGCTCTAAGCTTGG
	Rf1b	F	TCGATCGACCATGGCACGCCGCGTCGCTG
	Rf1b	R	TGCTCACCATGCGGTTGAAGCGGGACAC
	eGFP	F	AGTTCAAGGCATGGTGAGCAAGGGCGAGG
	eGFP	F*	CTTCAACCGCATGGTGAGCAAGGGCGAGG
	eGFP	R	ACGACGGCCAGTGCCAAGCTTGATCTAGTAACATAGATGACACCGC
StUBI10启动子组合	StUBI10	F	TATGACCATGATTACGAATTCCCAAGACAATTTCAGCTTAAAAAGT
	StUBI10	R	TGCTCACCATGCCTTGAACTGCTCTAAGCTTGG
	StUBI10	R*	CGCGGCGTGCCATGGATCCAAGCTTGAATCTGCAAATTC
	ATPγ	F	GCTTGGATCCATGGCAATGGCTGTTTTCCG
	ATPγ	R	TGCTCACCATGCCTTGAACTGCTCTAAGCTTGG
	Rf1b	F	GCTTGGATCCATGGCACGCCGCGTCGCTG
	Rf1b	R	TGCTCACCATGCGGTTGAAGCGGGACAC
	eGFP	F	AGTTCAAGGCATGGTGAGCAAGGGCGAGG
	eGFP	F*	CTTCAACCGCATGGTGAGCAAGGGCGAGG
	eGFP	R	ACGACGGCCAGTGCCAAGCTTGATCTAGTAACATAGATGACACCGC
AtUBI10启动子组合	AtUBI10	F	TATGACCATGATTACGAATTCATACGCTTCAATGCAGTGG
	AtUBI10	R	CCATTGCCATTGATAACTCTAGAATCTGTTAATCA
	AtUBI10	R*	GGCGTGCCATTGATAACTCTAGAATCTGTTAATCA
	ATPγ	F	AGAGTTATCAATGGCAATGGCTGTTTTCCG
	ATPγ	R	TGCTCACCATGCCTTGAACTGCTCTAAGCTTGG
	Rf1b	F	AGAGTTATCAATGGCACGCCGCGTCGCTG
	Rf1b	R	TGCTCACCATGCGGTTGAAGCGGGACAC
	eGFP	F	AGTTCAAGGCATGGTGAGCAAGGGCGAGG
	eGFP	F*	CTTCAACCGCATGGTGAGCAAGGGCGAGG
	eGFP	R	ACGACGGCCAGTGCCAAGCTTGATCTAGTAACATAGATGACACCGC
	P2300	F	GTTAGCTCACTCATTAGGCAC
	P2300	R	GCTGGCGTAATAGCGAAGAG

扩增片段Amplified fragment		引物序列Primer sequence（5'-3'）
2×35S启动子组合	2×35S	F	TATGACCATGATTACGAATTCGCTAGAGCAGCTTGCCAACATG
	2×35S	R	CCATTGCCATGGATCCAAGCTTGAAGAGAGAGAC
	2×35S	R*	GGCGTGCCATGGTCGATCGACAGATCTGCG
	ATPγ	F	TCGATCGACCATGGCAATGGCTGTTTTCCG
	ATPγ	R	TGCTCACCATGCCTTGAACTGCTCTAAGCTTGG
	Rf1b	F	TCGATCGACCATGGCACGCCGCGTCGCTG
	Rf1b	R	TGCTCACCATGCGGTTGAAGCGGGACAC
	eGFP	F	AGTTCAAGGCATGGTGAGCAAGGGCGAGG
	eGFP	F*	CTTCAACCGCATGGTGAGCAAGGGCGAGG
	eGFP	R	ACGACGGCCAGTGCCAAGCTTGATCTAGTAACATAGATGACACCGC
StUBI10启动子组合	StUBI10	F	TATGACCATGATTACGAATTCCCAAGACAATTTCAGCTTAAAAAGT
	StUBI10	R	TGCTCACCATGCCTTGAACTGCTCTAAGCTTGG
	StUBI10	R*	CGCGGCGTGCCATGGATCCAAGCTTGAATCTGCAAATTC
	ATPγ	F	GCTTGGATCCATGGCAATGGCTGTTTTCCG
	ATPγ	R	TGCTCACCATGCCTTGAACTGCTCTAAGCTTGG
	Rf1b	F	GCTTGGATCCATGGCACGCCGCGTCGCTG
	Rf1b	R	TGCTCACCATGCGGTTGAAGCGGGACAC
	eGFP	F	AGTTCAAGGCATGGTGAGCAAGGGCGAGG
	eGFP	F*	CTTCAACCGCATGGTGAGCAAGGGCGAGG
	eGFP	R	ACGACGGCCAGTGCCAAGCTTGATCTAGTAACATAGATGACACCGC
AtUBI10启动子组合	AtUBI10	F	TATGACCATGATTACGAATTCATACGCTTCAATGCAGTGG
	AtUBI10	R	CCATTGCCATTGATAACTCTAGAATCTGTTAATCA
	AtUBI10	R*	GGCGTGCCATTGATAACTCTAGAATCTGTTAATCA
	ATPγ	F	AGAGTTATCAATGGCAATGGCTGTTTTCCG
	ATPγ	R	TGCTCACCATGCCTTGAACTGCTCTAAGCTTGG
	Rf1b	F	AGAGTTATCAATGGCACGCCGCGTCGCTG
	Rf1b	R	TGCTCACCATGCGGTTGAAGCGGGACAC
	eGFP	F	AGTTCAAGGCATGGTGAGCAAGGGCGAGG
	eGFP	F*	CTTCAACCGCATGGTGAGCAAGGGCGAGG
	eGFP	R	ACGACGGCCAGTGCCAAGCTTGATCTAGTAACATAGATGACACCGC
	P2300	F	GTTAGCTCACTCATTAGGCAC
	P2300	R	GCTGGCGTAATAGCGAAGAG