AtMET1 Gene Cloning and Chemical-inducible Expression Analysis

LIANG Li-xiong; CHANG Ying-ying; GAO Ya-nan; WANG Yan-bo; ZHANG Wei-xi; SU Xiao-hua; ZHANG Bing-yu

Volume 29 Issue 6

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Article Navigation > Forest Research > 2016 > 29(6): 946-950

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AtMET1 Gene Cloning and Chemical-inducible Expression Analysis

1.
State Key Laboratory of Tree Genetics and Breeding, Key Laboratory of Tree Breeding and Cultivation of State Forestry Administration, Research Institute of Forestry Chinese Academy of Forestry, Beijing 100091, China

Received Date: 2015-08-28

Abstract

[Objective] In order to study the function of Arabidopsis thaliana methyltransferase gene AtMET1, 17-β-estradiol inducible plant expression vector pER8-MET1 was constructed, and its characters of inducible expression were detected. [Methods] The AtMET1 gene from Arabidopsis thaliana was used as the target gene, and ‘digestion-ligation’ method was applied for constructing the plant expression vector pER8-MET1, which could be induced by 17-β-estradiol. Then the vector was transferred to Agrobacterium tumefaciens LBA4404. The Agrobacterium strain containing pER8-GFP vector was injected into the leaves of Nicotiana tabacum, then the leaves were induced by 17-β-estradiol in different levels of concentrations and time durations. QPCR was performed to detect GFP gene expression, through which the optimum concentration and time duration were screened. The Agrobacterium strain containing pER8-MET1 vector was injected into the leaves of Nicotiana tabacum, then the leaves were induced by optimal concentration of 17-β-estradiol through different time durations. QPCR was performed to detect MET1 gene expression. [Results] The results of qPCR showed that 17-β-estradiol can effectively induce the expression of the target gene in pER8-MET1 and the optimal concentration of 17-β-estradiol is 50 μmol·L-1. The expression level of MET1 gene gradually increased with 17-β-estradiol treating time and reached the highest level at 12h. [Conclusion] The pER8-MET1 vector was successfully constructed. The construction of the plant expression vector pER8-MET1 lays a good foundation for the mechanism study on the relationship between DNA methylation and phenotype variation.
- pER8-MET1,
- 17-β-estradiol,
- chemical-inducible expression vector,
- transient expression

References

[1]	Lukens L N, Zhan S. The plant genome's methylation status and response to stress: implications for plant improvement[J]. Current Opinion in Plant Biology, 2007, 10(3): 317-322.
[2]	Jullien P E, Mosquna A, Ingouff M, et al. Retinoblastoma and its binding partner MSI1 control imprinting in Arabidopsis[J]. PLoS Biol, 2008, 6(8): e194.
[3]	Finnegan E J, Dennis E S. Isolation and identification by sequence homology of a putative cytosine methyltransferase from Arabidopsis thaliana[J]. Nucleic Acids Research, 1993, 21(10): 2383-2388.
[4]	Finnegan E J, Peacock W J, Dennis E S. Reduced DNA methylation in Arabidopsis thaliana results in abnormal plant development[J]. Proceedings of the National Academy of Sciences, 1996, 93(16): 8449-8454.
[5]	Kato M, Miura A, Bender J, et al. Role of CG and non-CG methylation in immobilization of transposons in Arabidopsis[J]. Current Biology, 2003, 13(5): 421-426.
[6]	López-Maury L, Marguerat S, Bähler J. Tuning gene expression to changing environments: from rapid responses to evolutionary adaptation[J]. Nature Reviews Genetics, 2008, 9(8): 583-593.
[7]	Kankel M W, Ramsey D E, Stokes T L, et al. Arabidopsis MET1 cytosine methyltransferase mutants[J]. Genetics, 2003, 163(3): 1109-1122.
[8]	Deleris A, Stroud H, Bernatavichute Y, et al. Loss of the DNA methyltransferase MET1 Induces H3K9 hypermethylation at PcG target genes and redistribution of H3K27 trimethylation to transposons in Arabidopsis thaliana[J]. PLoS Genetics, 2012, 8(11): e1003062.
[9]	Lira-Medeiros C F, Parisod C, Fernandes R A, et al. Epigenetic variation in mangrove plants occurring in contrasting natural environment[J]. PLoS One, 2010, 5(4): e10326.
[10]	Finnegan E J. Epialleles-a source of random variation in times of stress[J]. Current Opinion in Plant Biology, 2002, 5(2): 101-106.
[11]	Deleris A, Stroud H, Bernatavichute Y, et al. Loss of the DNA methyltransferase MET1 Induces H3K9 hypermethylation at PcG target genes and redistribution of H3K27 trimethylation to transposons in Arabidopsis thaliana[J]. PLoS Genetics, 2012, 8(11): e1003062.
[12]	庄晓英, 卢钢, 曹家树, 等. 化学诱导表达系统及其在植物中的应用[J]. 细胞生物学杂志, 2005, 27(4): 407-413.
[13]	Zuo J, Niu Q W, Chua N H. An estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plants[J]. The Plant Journal, 2000, 24(2): 265-273.
[14]	Martin L, Decourteix M, Badel E, et al. The zinc finger protein PtaZFP2 negatively controls stem growth and gene expression responsiveness to external mechanical loads in poplar[J]. New Phytologist, 2014, 203(1): 168-181.
[15]	Pfaffl M W, Horgan G W, Dempfle L. Relative expression software tool (REST©) for group-wise comparison and statistical analysis of relative expression results in real-time PCR[J]. Nucleic acids research, 2002, 30(9): e36-e36.
[16]	Sparkes I A, Runions J, Kearns A, et al. Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants[J]. Nature Protocols, 2006, 1(4): 2019-2025.
[17]	朱俊华, 竺晓平, 温孚江, 等. 马铃薯 Y 病毒衣壳蛋白基因片段长度对 RNA 介导抗病性的影响[J]. 中国科学: C 辑, 2004, 34(1): 23-30.
[18]	欧阳乐军, 黄真池, 沙月娥, 等. 植物病原菌诱导表达载体构建及在烟草中的瞬时表达研究[J]. 华北农学报, 2013, 28(4): 41-45.
[19]	吴英杰, 姜波, 张岩, 等. 农杆菌介导的烟草瞬时表达试验条件优化[J]. 东北林业大学学报, 2010, 38(9): 110-112.
[20]	谭小力, 诸葛锐军, 李冠英, 等. 农杆菌介导的油菜子叶瞬时表达[J]. 生物学杂志, 2013, 29(6): 93-96.

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

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AtMET1 Gene Cloning and Chemical-inducible Expression Analysis

1. State Key Laboratory of Tree Genetics and Breeding, Key Laboratory of Tree Breeding and Cultivation of State Forestry Administration, Research Institute of Forestry Chinese Academy of Forestry, Beijing 100091, China

Received Date: 2015-08-28

Abstract: [Objective] In order to study the function of Arabidopsis thaliana methyltransferase gene AtMET1, 17-β-estradiol inducible plant expression vector pER8-MET1 was constructed, and its characters of inducible expression were detected. [Methods] The AtMET1 gene from Arabidopsis thaliana was used as the target gene, and ‘digestion-ligation’ method was applied for constructing the plant expression vector pER8-MET1, which could be induced by 17-β-estradiol. Then the vector was transferred to Agrobacterium tumefaciens LBA4404. The Agrobacterium strain containing pER8-GFP vector was injected into the leaves of Nicotiana tabacum, then the leaves were induced by 17-β-estradiol in different levels of concentrations and time durations. QPCR was performed to detect GFP gene expression, through which the optimum concentration and time duration were screened. The Agrobacterium strain containing pER8-MET1 vector was injected into the leaves of Nicotiana tabacum, then the leaves were induced by optimal concentration of 17-β-estradiol through different time durations. QPCR was performed to detect MET1 gene expression. [Results] The results of qPCR showed that 17-β-estradiol can effectively induce the expression of the target gene in pER8-MET1 and the optimal concentration of 17-β-estradiol is 50 μmol·L-1. The expression level of MET1 gene gradually increased with 17-β-estradiol treating time and reached the highest level at 12h. [Conclusion] The pER8-MET1 vector was successfully constructed. The construction of the plant expression vector pER8-MET1 lays a good foundation for the mechanism study on the relationship between DNA methylation and phenotype variation.

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[1]	Lukens L N, Zhan S. The plant genome's methylation status and response to stress: implications for plant improvement[J]. Current Opinion in Plant Biology, 2007, 10(3): 317-322.
[2]	Jullien P E, Mosquna A, Ingouff M, et al. Retinoblastoma and its binding partner MSI1 control imprinting in Arabidopsis[J]. PLoS Biol, 2008, 6(8): e194.
[3]	Finnegan E J, Dennis E S. Isolation and identification by sequence homology of a putative cytosine methyltransferase from Arabidopsis thaliana[J]. Nucleic Acids Research, 1993, 21(10): 2383-2388.
[4]	Finnegan E J, Peacock W J, Dennis E S. Reduced DNA methylation in Arabidopsis thaliana results in abnormal plant development[J]. Proceedings of the National Academy of Sciences, 1996, 93(16): 8449-8454.
[5]	Kato M, Miura A, Bender J, et al. Role of CG and non-CG methylation in immobilization of transposons in Arabidopsis[J]. Current Biology, 2003, 13(5): 421-426.
[6]	López-Maury L, Marguerat S, Bähler J. Tuning gene expression to changing environments: from rapid responses to evolutionary adaptation[J]. Nature Reviews Genetics, 2008, 9(8): 583-593.
[7]	Kankel M W, Ramsey D E, Stokes T L, et al. Arabidopsis MET1 cytosine methyltransferase mutants[J]. Genetics, 2003, 163(3): 1109-1122.
[8]	Deleris A, Stroud H, Bernatavichute Y, et al. Loss of the DNA methyltransferase MET1 Induces H3K9 hypermethylation at PcG target genes and redistribution of H3K27 trimethylation to transposons in Arabidopsis thaliana[J]. PLoS Genetics, 2012, 8(11): e1003062.
[9]	Lira-Medeiros C F, Parisod C, Fernandes R A, et al. Epigenetic variation in mangrove plants occurring in contrasting natural environment[J]. PLoS One, 2010, 5(4): e10326.
[10]	Finnegan E J. Epialleles-a source of random variation in times of stress[J]. Current Opinion in Plant Biology, 2002, 5(2): 101-106.
[11]	Deleris A, Stroud H, Bernatavichute Y, et al. Loss of the DNA methyltransferase MET1 Induces H3K9 hypermethylation at PcG target genes and redistribution of H3K27 trimethylation to transposons in Arabidopsis thaliana[J]. PLoS Genetics, 2012, 8(11): e1003062.
[12]	庄晓英, 卢钢, 曹家树, 等. 化学诱导表达系统及其在植物中的应用[J]. 细胞生物学杂志, 2005, 27(4): 407-413.
[13]	Zuo J, Niu Q W, Chua N H. An estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plants[J]. The Plant Journal, 2000, 24(2): 265-273.
[14]	Martin L, Decourteix M, Badel E, et al. The zinc finger protein PtaZFP2 negatively controls stem growth and gene expression responsiveness to external mechanical loads in poplar[J]. New Phytologist, 2014, 203(1): 168-181.
[15]	Pfaffl M W, Horgan G W, Dempfle L. Relative expression software tool (REST©) for group-wise comparison and statistical analysis of relative expression results in real-time PCR[J]. Nucleic acids research, 2002, 30(9): e36-e36.
[16]	Sparkes I A, Runions J, Kearns A, et al. Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants[J]. Nature Protocols, 2006, 1(4): 2019-2025.
[17]	朱俊华, 竺晓平, 温孚江, 等. 马铃薯 Y 病毒衣壳蛋白基因片段长度对 RNA 介导抗病性的影响[J]. 中国科学: C 辑, 2004, 34(1): 23-30.
[18]	欧阳乐军, 黄真池, 沙月娥, 等. 植物病原菌诱导表达载体构建及在烟草中的瞬时表达研究[J]. 华北农学报, 2013, 28(4): 41-45.
[19]	吴英杰, 姜波, 张岩, 等. 农杆菌介导的烟草瞬时表达试验条件优化[J]. 东北林业大学学报, 2010, 38(9): 110-112.
[20]	谭小力, 诸葛锐军, 李冠英, 等. 农杆菌介导的油菜子叶瞬时表达[J]. 生物学杂志, 2013, 29(6): 93-96.

AtMET1 Gene Cloning and Chemical-inducible Expression Analysis

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通讯作者: 陈斌, bchen63@163.com

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AtMET1 Gene Cloning and Chemical-inducible Expression Analysis

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