Embryo Protein Extraction from Ginkgo biloba and Optimization of Two-Dimensional Electrophoresis

LI Jia; LU Xiu-jun; MEI Mei; SUN Xiao-mei; ZHANG Xiao-lin

Volume 29 Issue 3

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Article Navigation > Forest Research > 2016 > 29(3): 383-388

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Embryo Protein Extraction from Ginkgo biloba and Optimization of Two-Dimensional Electrophoresis

1.
College of Forestry, Shenyang Agricultural University, Shenyang 110161, Liaoning, China
2.
College of Horticulture, Shenyang Agricultural University, Shenyang 110161, Liaoning, China

Received Date: 2015-12-07

Abstract

[Objective] A two-dimensional (2-DE) electrophoresis system was explored for proteomic analysis of Ginkgo biloba embryo, aiming at laying a foundation for studying the differences in protein expression at different developmental stages.[Method] The 2-DE gels were involved with different protein extraction methods (direct extraction method, phenol extraction method, Tris-HCl method, Trizol extraction and TCA-acetone-phenol method), different concentrations of urea in lysis buffer (6 mol·L-1, 7 mol·L-1, 8 mol·L-1, and 9 mol·L-1), different protein loading dosages (1500 μg, 1650 μg, and 1800 μg) and different separation gel concentrations (10%, 12.5%, and 15%), to select the suitable condition.[Result] The optimized system includes the following steps:extracting total protein from G. biloba embryo by TCA-acetone-phenol method,dissolving proteins with lysis buffer containing 8 mol·L-1 urea,1650 μg of protein loading dosage and running SDS-PAGE with 12.5% gel concentration. Reproducible profiles with high resolution and clean background were obtained by this optimized two dimensional electrophoresis of embryonic total protein.[Conclusion] The optimized 2-DE electrophoresis system suitable for the separation of embryonic total protein from G. biloba was established, which is:extracting total protein by TCA-acetone-phenol method,dissolving proteins with lysis buffer containing 8 mol·L-1urea, 1650 μg of protein loading dosage and running SDS-PAGE with 12.5% gel concentration.
- Ginkgo biloba,
- embryo,
- protein,
- two-dimensional electrophoresis

References

[1]	郝明灼, 曹福亮, 汪贵斌, 等. 银杏杂交育种研究现状及展望[J]. 林业科技开发, 2005(06):8-11.
[2]	朱丽峰. 银杏在园林绿化中应用现状调查与分析[D]. 湖南农业大学, 2012.
[3]	江静, 尚富德, 李锁平. 银杏愈伤组织的诱导与激素优化组合研究[J]. 河南大学学报:自然科学版, 2015,30(3):51-54.
[4]	王雁, 杨义芳. 银杏叶的药理作用及其机制的研究进展[J]. 中国现代应用药学, 2001,18(1):1-3.
[5]	李然红, 于丽杰, 安文和, 等. 银杏种子繁殖技术研究[J]. 北方园艺, 2012,17期(17):151-152.
[6]	赵春香, 杨凤娟, 潘武扬, 等. 不同后熟条件对银杏种子萌发的影响[J]. 广东农业科学, 2005,03期(3):32-33.
[7]	芮海云. 银杏种子发育和后熟生理研究[J]. 廊坊师范学院学报:自然科学版, 2009(03):65-68.
[8]	杨玉珍, 李生平, 吴青霞, 等. 银杏种子萌发过程中蛋白质及3种氮代谢酶活性的变化[J]. 南京林业大学学报:自然科学版, 2006,30(04):119-122.
[9]	王亚茹, 李云, 孙宇涵, 等. 银杏小孢子蛋白提取及双向电泳技术优化[J]. 西南林学院学报, 2010(03):45-49.
[10]	杨妮娜, 崔彬彬, 孙宇涵, 等. 银杏小孢子叶球蛋白提取和双向电泳体系优化(Ⅱ)[J]. 东北林业大学学报, 2011(08):55-57.
[11]	李生平. 银杏种子萌发过程中贮藏物质代谢机理的研究[D]. 南京林业大学, 2004.
[12]	M C A, A K, T P, et al. Control of early seed development.[J]. Annual Review of Cell and Developmental Biology, 2001,17.
[13]	A S P, A L B. The development of endosperm in grasses.[J]. Plant Physiology, 2009,149(1).
[14]	Chao H, Dongli H, Ming L, et al. In-depth proteomic analysis of rice embryo reveals its important roles in seed germination.[J]. Plant & Cell Physiology, 2014,55(10):1826-1847.
[15]	陆秀君, 刘月洋, 陈晓旭, 等. 天女木兰种子后熟期间的生理生化变化[J]. 北京林业大学学报, 2009(06):164-168.
[16]	杨国会,孙立梅,邢力,等.不同层积处理方法对东北红豆杉种子后熟过程中贮藏物质的影响[J]. 北方园艺, 2013(14):82-85.
[17]	韩春艳. 木兰科几种植物种子休眠和种质资源保存的研究[D]. 中国科学院昆明植物研究所, 2008.
[18]	张鹏, 沈海龙.不同发育时期水曲柳种子的形态生理变化及其层积处理后的萌发效应[J]. 植物生理学报, 2010,第2期(02):125-130.
[19]	郭广芳, 董坤, 高利艳, 等.小麦根蛋白提取与双向电泳方法的优化与应用[J]. 植物遗传资源学报, 2010(02):244-248.
[20]	张晓林,陆秀君,马蓓蓓,等. 天女木兰种子蛋白双向电泳体系的建立[J]. 西北农林科技大学学报:自然科学版, 2012(08):207-214.
[21]	曾广娟, 李春敏, 张新忠, 等. 适于SDS-PAGE分析的苹果叶片蛋白质提取方法[J]. 华北农学报, 2009(02):75-78.
[22]	M B M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.[J]. Analytical Biochemistry, 1976,72.
[23]	Wongpia A, Mahatheeranont S, Lomthaisong K, et al. Evaluation of Sample Preparation Methods from Rice Seeds and Seedlings Suitable for Two-Dimensional Gel Electrophoresis[J]. Applied Biochemistry and Biotechnology, 2015,175(2).
[24]	郭广芳, 董坤, 高利艳, 等.小麦根蛋白提取与双向电泳方法的优化与应用[J]. 植物遗传资源学报, 2010(02):244-248.
[25]	陈蕊红, 张改生, 刘卫, 等.小麦花药蛋白质组双向电泳技术体系的优化[J]. 核农学报, 2008(04):404-409.

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通讯作者: 陈斌, bchen63@163.com

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沈阳化工大学材料科学与工程学院沈阳 110142

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Embryo Protein Extraction from Ginkgo biloba and Optimization of Two-Dimensional Electrophoresis

1. College of Forestry, Shenyang Agricultural University, Shenyang 110161, Liaoning, China

2. College of Horticulture, Shenyang Agricultural University, Shenyang 110161, Liaoning, China

Received Date: 2015-12-07

Abstract: [Objective] A two-dimensional (2-DE) electrophoresis system was explored for proteomic analysis of Ginkgo biloba embryo, aiming at laying a foundation for studying the differences in protein expression at different developmental stages.[Method] The 2-DE gels were involved with different protein extraction methods (direct extraction method, phenol extraction method, Tris-HCl method, Trizol extraction and TCA-acetone-phenol method), different concentrations of urea in lysis buffer (6 mol·L-1, 7 mol·L-1, 8 mol·L-1, and 9 mol·L-1), different protein loading dosages (1500 μg, 1650 μg, and 1800 μg) and different separation gel concentrations (10%, 12.5%, and 15%), to select the suitable condition.[Result] The optimized system includes the following steps:extracting total protein from G. biloba embryo by TCA-acetone-phenol method,dissolving proteins with lysis buffer containing 8 mol·L-1 urea,1650 μg of protein loading dosage and running SDS-PAGE with 12.5% gel concentration. Reproducible profiles with high resolution and clean background were obtained by this optimized two dimensional electrophoresis of embryonic total protein.[Conclusion] The optimized 2-DE electrophoresis system suitable for the separation of embryonic total protein from G. biloba was established, which is:extracting total protein by TCA-acetone-phenol method,dissolving proteins with lysis buffer containing 8 mol·L-1urea, 1650 μg of protein loading dosage and running SDS-PAGE with 12.5% gel concentration.

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[1]	郝明灼, 曹福亮, 汪贵斌, 等. 银杏杂交育种研究现状及展望[J]. 林业科技开发, 2005(06):8-11.
[2]	朱丽峰. 银杏在园林绿化中应用现状调查与分析[D]. 湖南农业大学, 2012.
[3]	江静, 尚富德, 李锁平. 银杏愈伤组织的诱导与激素优化组合研究[J]. 河南大学学报:自然科学版, 2015,30(3):51-54.
[4]	王雁, 杨义芳. 银杏叶的药理作用及其机制的研究进展[J]. 中国现代应用药学, 2001,18(1):1-3.
[5]	李然红, 于丽杰, 安文和, 等. 银杏种子繁殖技术研究[J]. 北方园艺, 2012,17期(17):151-152.
[6]	赵春香, 杨凤娟, 潘武扬, 等. 不同后熟条件对银杏种子萌发的影响[J]. 广东农业科学, 2005,03期(3):32-33.
[7]	芮海云. 银杏种子发育和后熟生理研究[J]. 廊坊师范学院学报:自然科学版, 2009(03):65-68.
[8]	杨玉珍, 李生平, 吴青霞, 等. 银杏种子萌发过程中蛋白质及3种氮代谢酶活性的变化[J]. 南京林业大学学报:自然科学版, 2006,30(04):119-122.
[9]	王亚茹, 李云, 孙宇涵, 等. 银杏小孢子蛋白提取及双向电泳技术优化[J]. 西南林学院学报, 2010(03):45-49.
[10]	杨妮娜, 崔彬彬, 孙宇涵, 等. 银杏小孢子叶球蛋白提取和双向电泳体系优化(Ⅱ)[J]. 东北林业大学学报, 2011(08):55-57.
[11]	李生平. 银杏种子萌发过程中贮藏物质代谢机理的研究[D]. 南京林业大学, 2004.
[12]	M C A, A K, T P, et al. Control of early seed development.[J]. Annual Review of Cell and Developmental Biology, 2001,17.
[13]	A S P, A L B. The development of endosperm in grasses.[J]. Plant Physiology, 2009,149(1).
[14]	Chao H, Dongli H, Ming L, et al. In-depth proteomic analysis of rice embryo reveals its important roles in seed germination.[J]. Plant & Cell Physiology, 2014,55(10):1826-1847.
[15]	陆秀君, 刘月洋, 陈晓旭, 等. 天女木兰种子后熟期间的生理生化变化[J]. 北京林业大学学报, 2009(06):164-168.
[16]	杨国会,孙立梅,邢力,等.不同层积处理方法对东北红豆杉种子后熟过程中贮藏物质的影响[J]. 北方园艺, 2013(14):82-85.
[17]	韩春艳. 木兰科几种植物种子休眠和种质资源保存的研究[D]. 中国科学院昆明植物研究所, 2008.
[18]	张鹏, 沈海龙.不同发育时期水曲柳种子的形态生理变化及其层积处理后的萌发效应[J]. 植物生理学报, 2010,第2期(02):125-130.
[19]	郭广芳, 董坤, 高利艳, 等.小麦根蛋白提取与双向电泳方法的优化与应用[J]. 植物遗传资源学报, 2010(02):244-248.
[20]	张晓林,陆秀君,马蓓蓓,等. 天女木兰种子蛋白双向电泳体系的建立[J]. 西北农林科技大学学报:自然科学版, 2012(08):207-214.
[21]	曾广娟, 李春敏, 张新忠, 等. 适于SDS-PAGE分析的苹果叶片蛋白质提取方法[J]. 华北农学报, 2009(02):75-78.
[22]	M B M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.[J]. Analytical Biochemistry, 1976,72.
[23]	Wongpia A, Mahatheeranont S, Lomthaisong K, et al. Evaluation of Sample Preparation Methods from Rice Seeds and Seedlings Suitable for Two-Dimensional Gel Electrophoresis[J]. Applied Biochemistry and Biotechnology, 2015,175(2).
[24]	郭广芳, 董坤, 高利艳, 等.小麦根蛋白提取与双向电泳方法的优化与应用[J]. 植物遗传资源学报, 2010(02):244-248.
[25]	陈蕊红, 张改生, 刘卫, 等.小麦花药蛋白质组双向电泳技术体系的优化[J]. 核农学报, 2008(04):404-409.

Embryo Protein Extraction from Ginkgo biloba and Optimization of Two-Dimensional Electrophoresis

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通讯作者: 陈斌, bchen63@163.com

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Embryo Protein Extraction from Ginkgo biloba and Optimization of Two-Dimensional Electrophoresis

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