适用于花叶唐竹叶片总蛋白的双向电泳体系的建立
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摘要
本研究以花叶唐竹二年生苗木的健康叶片为材料,采用3种不同的蛋白质提取方法(酚法, TCA-丙酮法, TCA-丙酮/酚法)、不同的上样量、不同p H值IPG胶条及不同等电聚焦方案等因素进行筛选和优化,以期获得最佳的花叶唐竹叶片总蛋白的提取方法,并建立适用、高效的花叶唐竹蛋白质双向凝胶电泳技术体系。结果表明,选择TCA-丙酮/酚法的蛋白得率高、纯度高,选择17 cm pH 5～8的线性IPG胶条,450μg的上样量以及60 000 Vhs的等电聚焦强度进行双向电泳,共检测到726个蛋白点,整体分布均匀,分离效果较好。本研究初步建立了花叶唐竹蛋白质双向电泳体系,有助于后续研究叶片呈色机理提供技术帮助。
In order to obtain the best extraction method of total protein of Sinobambusa tootsik and establish a suitable and efficient two-dimensional electrophoresis(2-DE) system for Sinobambusa tootsik proteomics analysis,the healthy leaves of biennial seedlings of Sinobambusa tootsik were used as materials in this study. different sample amount, different p H value IPG strips and different electrofocusing schemes were selected and optimized.Three protein extraction methods(phenol method, TCA-acetone precipitation method, and the TCA-acetone/phenol method), the pH range of IPG strips and isoelectric focusing programs were used for screening and optimization.The results showed that the TCA-acetone/phenol method selected could get a high purity and a high extraction rate. The effect of two-dimensional electrophoresis was excellent when conducted with immobilized 17 cm pH 5～8 IPG strips and 450 μg sample that 726 protein spots were detected, resulting in good separating effect and well distribution. This experiment preliminary established the 2-DE protocol of S. tootsik f. luteoloalbostriata leaves which would be beneficial for the further study of variegated leaves development mechanism.
In order to obtain the best extraction method of total protein of Sinobambusa tootsik and establish a suitable and efficient two-dimensional electrophoresis(2-DE) system for Sinobambusa tootsik proteomics analysis,the healthy leaves of biennial seedlings of Sinobambusa tootsik were used as materials in this study. different sample amount, different p H value IPG strips and different electrofocusing schemes were selected and optimized.Three protein extraction methods(phenol method, TCA-acetone precipitation method, and the TCA-acetone/phenol method), the pH range of IPG strips and isoelectric focusing programs were used for screening and optimization.The results showed that the TCA-acetone/phenol method selected could get a high purity and a high extraction rate. The effect of two-dimensional electrophoresis was excellent when conducted with immobilized 17 cm pH 5～8 IPG strips and 450 μg sample that 726 protein spots were detected, resulting in good separating effect and well distribution. This experiment preliminary established the 2-DE protocol of S. tootsik f. luteoloalbostriata leaves which would be beneficial for the further study of variegated leaves development mechanism.
引文
Alam I.,Sharmin S.A.,Kim K.H.,Lee J.J.,and Lee B.H.,2013,An improved plant leaf protein extraction method for high sesolution two-dimensional polyacrylamide gel electrophoresis and comparative proteomics,Biotech.Histochem.,88(2):61-75
Bodzon-Kulakowska A.,Bierczynska-Krzysik A.,Dylag T.,Drabik A.,Suder P.,Noga M.,Jarzebinska J.,and Silberring J.,2007,Methods for samples preparation in proteomic research,J.Chromatogr.B,849(1-2):1-31
Bradford M.M.,1976,A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding,Anal.Biochem.,72(S1-2):248-254
Chen J.Y.,Guo B.F.,He F.L.,Qu C.H.,Yin K.X.,Yang H.D.,and Zhao C.S.,2010,The comparison of protein extraction methods of plant for two-dimensional electrophoresis,Zhong guo Nongxue Tongbao(Chinese Agricultural Science Bulletin),26(23):97-100(陈晶瑜,郭宝峰,何付丽,曲春鹤,尹克鑫,杨洪达,赵长山,2010,适合双向电泳的植物全蛋白提取方法比较,中国农学通报,26(23):97-100)
Chen L.Y.,Lai J.L.,He T.Y.,Rong J.D.,Tarin M.W.K.,and Zheng Y.S.,2018,Differences in photosynthesis of variegated temple bamboo leaves with various levels of variegation are related to chlorophyll biosynthesis and chloroplast development,J.Amer.Soci.Hort.Sci.,143(2):144-153
Chen W.J.,He H.Y.,Deng Q.J.,and Peng F.R.,2017,The comparison on methods for determing different proteins and construction of protein two-dimensional electrophoresis system for Carya illinoensis,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(5):1874-1880(陈文静,何海洋,邓秋菊,彭方仁,2017,薄壳山核桃蛋白质测定方法比较以及双向电泳体系的建立,分子植物育种,15(5):1874-1880)
Corthals G.L.,Wasinger V.C.,Hochstrasser D.F.,and Sanshez J.C.,2000,The dynamic range of protein expression:a challenge for proteomic research,Electrophoresis,21(6):1104-1115
Dong H.X.,2010,Proteome analysis of chlorophyll-deficient mutant rice at the seedling stage,Dissertation for Ph.D.,Huazhong Agricultural University,Supervisor:Zeng H.L.,pp.14-26(董红霞,2010,水稻苗期叶色突变体的蛋白质组分析,博士学位论文,华中农业大学,导师:曾汉来,pp.14-26)
Fang X.M.,Jia J.L.,Tan J.Z.,Kan X.Q.,Liu Y.Y.,Chi X.J.,and Hirano H.,2009,Analysis of differential proteome of a leaf-variegated mutant cty-sm in mulberry(Morus L.),Canye Kexue(Acta Sericologica Sinica),35(1):1-5(方向明,贾俊丽,谈建中,阚雪芹,刘艳艳,池旭娟,平野久,2009,桑树斑状叶色突变体Cty-Sm差异蛋白质组的分析,蚕业科学,35(1):1-5)
Giavalisco P.,Nopdhoff E.,Lehrach H.,Gobom J.,and Klose J.,2003,Extraction of proteins from plant tissues for two-dimensional electrophoresis analysis,Electrophoresis,24(1-2):207-216
Granier F.,1988,Extraction of plant proteins for two-dimensional electrophoresis,Electrophoresis,9(11):712-718
Li F.,Xu Q.F.,Zang X.P.,Lai Y.Y.,Cheng W.S.,Xu Y.P.,and Cai X.Z.,2010,Establishment of two-dimensional electrophoresis system of tomato cotyledons,Yuanyi Xuebao(Acta Horticulturae Sinica),37(4):661-668(黎飞,徐秋芳,臧宪朋,赖亿玉,程维舜,徐幼平,蔡新忠,2010,番茄子叶总蛋白双向电泳体系的建立,园艺学报,37(4):661-668)
Liang W.Y.,Wang X.,Jiao G.F.,Zhang X.J.,and Chen W.,2009,Establishment and optimization of two-dimensional gel electrophoresis for proteome of Nostoc flagelliforme,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),29(8):1550-1556(梁文裕,王星,焦广飞,张喜娟,陈伟,2009,发菜蛋白质组双向电泳技术的建立及优化,西北植物学报,29(8):1550-1556)
Lin J.K.,Zhen J.G.,Yuan M.,Zhang X.Q.,and Wang F.,2003,The improvement of isolation and the method of two-dimensional electrophoresis of protein from tea plant,J.Tea Sci.,23(1):16-20
Liu M.Y.,Lu Y.,Zhao J.J.,Wang Y.H.,and Shen S.X.,2014,HRM identification and chlorophyll fluorescence characteristics on leaf color mutants in Chinese cabbage,Yuanyi Xuebao(Acta Horticulturae Sinica),41(11):2215-2224(刘梦洋,卢银,赵建军,王彦华,申书兴,2014,大白菜叶色突变体的HRM鉴定及叶绿素荧光参数分析,园艺学报,41(11):2215-2224)
Liu X.L.,Yang X.Y.,Yu H.J.,Jiang W.J.,Zhao W.C.,Wang Y.,and Zhang X.M.,2016,Establishment of two-dimensional electrophoresis system suitable for cucumber leave,root and fruit proteins,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(3):721-727(刘晓林,杨学勇,余宏军,蒋卫杰,赵文超,王燕,张晓孟,2016,适用于黄瓜叶片、根系和果实总蛋白的双向电泳体系的建立,基因组学与应用生物学,35(3):721-727)
Méchin V.,Consoli L.,Le Guilloux M.,and Damerval C.,2003,An efficient soluilization buffer for plant proteins focused in immobilized pH gradients,Proteomics,3:1299-1302
Pawlowski T.A.,2007,Proteomics of European beech(Fagus sylvatica L.)seed dormancy breaking:influence of abscisic and gibberellic acids,Proteomics,7(13):2246-2257
Rabilloud T.,and Lelong C.,2011,Two-dimensional gel electrophoresis in proteomics:a tutorial,Journal of Proteomics,74(10):1829-1841
SalgovicováI.,LojanováZ.,and Hudák J.,2007,Plastid structure and chlorophyll content in leaf primordia of onion bulbs,J.Plant Physiol.,164(1):19-22
Stockinger E.J.,and Walling L.L.,1994,A chlorophyll a/b-binding protein gene from soybean(Glycine max[L.]Merr.),Plant Physiol.,104(4):1475-1476
Wang W.,Vignani R.,Scali M.,and Mauro C.,2006,A universal and rapid protocol for protein extraction from recalcitrant plant tissues for proteomic analysis,Electrophoresis,27(13):2782-2786
Wildgruber R.,Harder A.,Obermaier C.,Boguth G.,Weiss W.,Fey S.J.,Larsen P.M.,and G觟rg A.,2000,Towards higher resolution:two-dimensional electrophoresis of Saccharomyces cerevisiae proteins using overlapping narrow immobilized pH gradients,Electrophoresis,21(13):2610-2616
Xu B.Q.,An M.M.,Jiang K.Y.,Xu L.L.,Yang H.Y.,and Zhou M.B.,2015,Cloning and functional analysis of the psbDgene in Pseudosasa japonica f.akebonosuji,Zhejiang Nonglin Daxue Xuebao(Journal of Zhejiang A&F University),32(4):557-565(许冰清,安苗苗,姜可以,徐丽丽,杨海芸,周明兵,2015,花叶矢竹叶绿体psbD基因的克隆与功能分析,浙江农林大学学报,32(4):557-565)
Xu L.,2012,Research and analysis of differential proteins from tomato yellow leaf mutant,Thesis for M.S.,Northeast Agriculture University,Supervisor:Li J.F.,pp.45(徐磊,2012,番茄叶色黄化突变体差异蛋白研究及分析,硕士学位论文,东北农业大学,导师:李景富,pp.45)
Zhang H.T.,Li J.J.,Yoo J.H.,Yoo S.C.,Cho S.H.,Koh H.J.,Seo H.S.,and Paek N.C.,2006,Rice Chlorina-1 and Chlorina-9encode ChlD and ChlI subunits of Mg-chelatase,a key enzyme for chlorophyll biosynthesis and chloroplast development,Plant Mol.Biol.,62(3):325-337
Zhang S.J.,Wang Q.,Liu Q.C.,Zhao H.,Zhao R.,and Wang Y.Q.,2016,Establishment of two-dimensional electrophoresis system for buds of welsh onion,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(2):403-409(张思佳,王清,刘倩纯,赵泓,赵瑞,王永勤,2016,大葱花蕾总蛋白双向电泳体系的建立,分子植物育种,14(2):403-409)
Bodzon-Kulakowska A.,Bierczynska-Krzysik A.,Dylag T.,Drabik A.,Suder P.,Noga M.,Jarzebinska J.,and Silberring J.,2007,Methods for samples preparation in proteomic research,J.Chromatogr.B,849(1-2):1-31
Bradford M.M.,1976,A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding,Anal.Biochem.,72(S1-2):248-254
Chen J.Y.,Guo B.F.,He F.L.,Qu C.H.,Yin K.X.,Yang H.D.,and Zhao C.S.,2010,The comparison of protein extraction methods of plant for two-dimensional electrophoresis,Zhong guo Nongxue Tongbao(Chinese Agricultural Science Bulletin),26(23):97-100(陈晶瑜,郭宝峰,何付丽,曲春鹤,尹克鑫,杨洪达,赵长山,2010,适合双向电泳的植物全蛋白提取方法比较,中国农学通报,26(23):97-100)
Chen L.Y.,Lai J.L.,He T.Y.,Rong J.D.,Tarin M.W.K.,and Zheng Y.S.,2018,Differences in photosynthesis of variegated temple bamboo leaves with various levels of variegation are related to chlorophyll biosynthesis and chloroplast development,J.Amer.Soci.Hort.Sci.,143(2):144-153
Chen W.J.,He H.Y.,Deng Q.J.,and Peng F.R.,2017,The comparison on methods for determing different proteins and construction of protein two-dimensional electrophoresis system for Carya illinoensis,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(5):1874-1880(陈文静,何海洋,邓秋菊,彭方仁,2017,薄壳山核桃蛋白质测定方法比较以及双向电泳体系的建立,分子植物育种,15(5):1874-1880)
Corthals G.L.,Wasinger V.C.,Hochstrasser D.F.,and Sanshez J.C.,2000,The dynamic range of protein expression:a challenge for proteomic research,Electrophoresis,21(6):1104-1115
Dong H.X.,2010,Proteome analysis of chlorophyll-deficient mutant rice at the seedling stage,Dissertation for Ph.D.,Huazhong Agricultural University,Supervisor:Zeng H.L.,pp.14-26(董红霞,2010,水稻苗期叶色突变体的蛋白质组分析,博士学位论文,华中农业大学,导师:曾汉来,pp.14-26)
Fang X.M.,Jia J.L.,Tan J.Z.,Kan X.Q.,Liu Y.Y.,Chi X.J.,and Hirano H.,2009,Analysis of differential proteome of a leaf-variegated mutant cty-sm in mulberry(Morus L.),Canye Kexue(Acta Sericologica Sinica),35(1):1-5(方向明,贾俊丽,谈建中,阚雪芹,刘艳艳,池旭娟,平野久,2009,桑树斑状叶色突变体Cty-Sm差异蛋白质组的分析,蚕业科学,35(1):1-5)
Giavalisco P.,Nopdhoff E.,Lehrach H.,Gobom J.,and Klose J.,2003,Extraction of proteins from plant tissues for two-dimensional electrophoresis analysis,Electrophoresis,24(1-2):207-216
Granier F.,1988,Extraction of plant proteins for two-dimensional electrophoresis,Electrophoresis,9(11):712-718
Li F.,Xu Q.F.,Zang X.P.,Lai Y.Y.,Cheng W.S.,Xu Y.P.,and Cai X.Z.,2010,Establishment of two-dimensional electrophoresis system of tomato cotyledons,Yuanyi Xuebao(Acta Horticulturae Sinica),37(4):661-668(黎飞,徐秋芳,臧宪朋,赖亿玉,程维舜,徐幼平,蔡新忠,2010,番茄子叶总蛋白双向电泳体系的建立,园艺学报,37(4):661-668)
Liang W.Y.,Wang X.,Jiao G.F.,Zhang X.J.,and Chen W.,2009,Establishment and optimization of two-dimensional gel electrophoresis for proteome of Nostoc flagelliforme,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),29(8):1550-1556(梁文裕,王星,焦广飞,张喜娟,陈伟,2009,发菜蛋白质组双向电泳技术的建立及优化,西北植物学报,29(8):1550-1556)
Lin J.K.,Zhen J.G.,Yuan M.,Zhang X.Q.,and Wang F.,2003,The improvement of isolation and the method of two-dimensional electrophoresis of protein from tea plant,J.Tea Sci.,23(1):16-20
Liu M.Y.,Lu Y.,Zhao J.J.,Wang Y.H.,and Shen S.X.,2014,HRM identification and chlorophyll fluorescence characteristics on leaf color mutants in Chinese cabbage,Yuanyi Xuebao(Acta Horticulturae Sinica),41(11):2215-2224(刘梦洋,卢银,赵建军,王彦华,申书兴,2014,大白菜叶色突变体的HRM鉴定及叶绿素荧光参数分析,园艺学报,41(11):2215-2224)
Liu X.L.,Yang X.Y.,Yu H.J.,Jiang W.J.,Zhao W.C.,Wang Y.,and Zhang X.M.,2016,Establishment of two-dimensional electrophoresis system suitable for cucumber leave,root and fruit proteins,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(3):721-727(刘晓林,杨学勇,余宏军,蒋卫杰,赵文超,王燕,张晓孟,2016,适用于黄瓜叶片、根系和果实总蛋白的双向电泳体系的建立,基因组学与应用生物学,35(3):721-727)
Méchin V.,Consoli L.,Le Guilloux M.,and Damerval C.,2003,An efficient soluilization buffer for plant proteins focused in immobilized pH gradients,Proteomics,3:1299-1302
Pawlowski T.A.,2007,Proteomics of European beech(Fagus sylvatica L.)seed dormancy breaking:influence of abscisic and gibberellic acids,Proteomics,7(13):2246-2257
Rabilloud T.,and Lelong C.,2011,Two-dimensional gel electrophoresis in proteomics:a tutorial,Journal of Proteomics,74(10):1829-1841
SalgovicováI.,LojanováZ.,and Hudák J.,2007,Plastid structure and chlorophyll content in leaf primordia of onion bulbs,J.Plant Physiol.,164(1):19-22
Stockinger E.J.,and Walling L.L.,1994,A chlorophyll a/b-binding protein gene from soybean(Glycine max[L.]Merr.),Plant Physiol.,104(4):1475-1476
Wang W.,Vignani R.,Scali M.,and Mauro C.,2006,A universal and rapid protocol for protein extraction from recalcitrant plant tissues for proteomic analysis,Electrophoresis,27(13):2782-2786
Wildgruber R.,Harder A.,Obermaier C.,Boguth G.,Weiss W.,Fey S.J.,Larsen P.M.,and G觟rg A.,2000,Towards higher resolution:two-dimensional electrophoresis of Saccharomyces cerevisiae proteins using overlapping narrow immobilized pH gradients,Electrophoresis,21(13):2610-2616
Xu B.Q.,An M.M.,Jiang K.Y.,Xu L.L.,Yang H.Y.,and Zhou M.B.,2015,Cloning and functional analysis of the psbDgene in Pseudosasa japonica f.akebonosuji,Zhejiang Nonglin Daxue Xuebao(Journal of Zhejiang A&F University),32(4):557-565(许冰清,安苗苗,姜可以,徐丽丽,杨海芸,周明兵,2015,花叶矢竹叶绿体psbD基因的克隆与功能分析,浙江农林大学学报,32(4):557-565)
Xu L.,2012,Research and analysis of differential proteins from tomato yellow leaf mutant,Thesis for M.S.,Northeast Agriculture University,Supervisor:Li J.F.,pp.45(徐磊,2012,番茄叶色黄化突变体差异蛋白研究及分析,硕士学位论文,东北农业大学,导师:李景富,pp.45)
Zhang H.T.,Li J.J.,Yoo J.H.,Yoo S.C.,Cho S.H.,Koh H.J.,Seo H.S.,and Paek N.C.,2006,Rice Chlorina-1 and Chlorina-9encode ChlD and ChlI subunits of Mg-chelatase,a key enzyme for chlorophyll biosynthesis and chloroplast development,Plant Mol.Biol.,62(3):325-337
Zhang S.J.,Wang Q.,Liu Q.C.,Zhao H.,Zhao R.,and Wang Y.Q.,2016,Establishment of two-dimensional electrophoresis system for buds of welsh onion,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(2):403-409(张思佳,王清,刘倩纯,赵泓,赵瑞,王永勤,2016,大葱花蕾总蛋白双向电泳体系的建立,分子植物育种,14(2):403-409)