华南8号木薯块根形成期叶绿体蛋白质组学分析
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摘要
以‘华南8号’木薯块根形成期叶绿体为材料,采用改进酚抽法提取总蛋白,通过一维和二维电泳,经Image Master分析,获得了木薯叶绿体的蛋白质表达谱,发现(397±31)个蛋白点;挖取2-DE凝胶上相对丰度较高的蛋白点275个,利用改进的酶解方法进行胶内酶解,经MALDI-TOF MS质谱鉴定,获得208个蛋白点,对应143种蛋白质,这些蛋白主要参与光合作用、光合生物碳固定、氧化还原调节、氨基酸代谢、蛋白质代谢和转运等途径。结果为后期从蛋白质组水平深入研究木薯叶绿体高光效机制提供一定参考。
The improved phenol method was used for total protein extraction from the chloroplast of forming stage tuberous root in cassava cultivar ‘South China 8'(SC8), and these proteins expressed profile were determined with1-DE and 2-DE. The obtained typical 2-DE gels were analyzed by Image Master software, and generated(397±31)protein spots. Two hundred and seventy five protein points with relatively high abundance were harvested and enzymolized using the improved enzyme solution method. Two hundred and eight proteins points were finally identified by MALDI-TOF MS, corresponding to 143 proteins, which were involved in photosynthesis, carbon fixation, redox balance regulation, amino acid metabolism, protein metabolism and transport, and so forth. This study acquired the information of cassava chloroplast proteins during the tuberous root formation, preliminarily analyzed these proteins' function, and provided an important fundamental technology for further proteomic research on the high photosynthetic efficiency mechanism of cassava cultivar.
The improved phenol method was used for total protein extraction from the chloroplast of forming stage tuberous root in cassava cultivar ‘South China 8'(SC8), and these proteins expressed profile were determined with1-DE and 2-DE. The obtained typical 2-DE gels were analyzed by Image Master software, and generated(397±31)protein spots. Two hundred and seventy five protein points with relatively high abundance were harvested and enzymolized using the improved enzyme solution method. Two hundred and eight proteins points were finally identified by MALDI-TOF MS, corresponding to 143 proteins, which were involved in photosynthesis, carbon fixation, redox balance regulation, amino acid metabolism, protein metabolism and transport, and so forth. This study acquired the information of cassava chloroplast proteins during the tuberous root formation, preliminarily analyzed these proteins' function, and provided an important fundamental technology for further proteomic research on the high photosynthetic efficiency mechanism of cassava cultivar.
引文
[1]El-Sharkawy M A.Cassava biology and physiology[J].Plant Molecular Biology,2004,56(4):481-501.
[2]张鹏,安冬,马秋香,等.木薯分子育种中若干基本科学问题的思考与研究进展[J].中国科学,2013,43(12):1 082-1 089.
[3]贺庭琪,徐兵强,郭安平,等.不同品种木薯叶绿体比较蛋白质组学初步研究[J].热带作物学报,2013,34(6):1 090-1 097.
[4]张根连,范术丽,宋美珍,等.植物蛋白质组学技术研究进展[J].生物技术通报,2011,7:26-30.
[5]Frank G,Pressman E,Ophir R,et al.Transcriptional profiling of maturing tomato(Solanum lycopersicum L.)microspores reveals the involvement of heat shock proteins,ROS scavengers,hormones,and sugars in the heat stress response[J].Journal of Experimental Botany,2009,60(13):3 891-3 908.
[6]Baginsky S,Gruissem W.Chloroplast proteomics:potentials and challenges[J].Journal of Experimental Botany,2004,55(400):1 213-1 220.
[7]Jorrín J,Maldonado A,Castillejo M.Plant proteome analysis:a 2006 update[J].Proteomics,2007,7(16):2 947-2 962.
[8]Schroder W,Kieselbach T.Update on chloroplast proteomics[J].Photosynthesis Research,2003,78:181-193.
[9]Fan P X,Wang X C,Kuang T Y,et al.An efficient method for the extraction of chloroplast proteins compatible for 2-DE and MS analysis[J].Electrophoresis,2009,30(17):3 024-3 033.
[10]Saravanan R S,Rose J C.A critical evaluation of sample extraction techniques for enhanced proteomic analysis of recalcitrant plant tissues[J].Proteomics,2004,4:2 522-2 532.
[11]Wang X C,Li X F,Han H P,et al.A protein extraction method compatible with proteomic analysis for euhalophyte Salicornia europaea L[J].Electrophoresis,2007,28(21):3 976-3 987.
[12]Bradford M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding[J].Analytical Biochemistry,1976,72:248-254.
[13]Laemmli U K.Cleavage of structural proteins during the assembly of the head of bacteriophage T4[J].Nature,1970,227(5 259):680-685.
[14]Wang X C,Wang D Y,Wang D,et al.Systematic comparison of technical details in CBB methods and development of a sensitive GAP stain for comparative proteomic analysis[J].Electrophoresis,2012,33(2):296-306.
[15]王旭初,范鹏祥,李银心.一种适用于质谱分析的简化胶内酶解方法[J].植物生理与分子生物学报,2007,33:449-455.
[16]Shen S H,Jing Y X,Kuang T Y.Proteomics approach to identify wound-response related proteins from rice leaf sheath[J].Proteomics,2003,3(4):527-535.
[17]Arun Goysl,Thomas Betsche,Tolbert N E.Isolation of Intact Chloroplasts from Dunaliella Tertiolecta[J].Plant Physiol,1988,88(2):543-546.
[18]王力敏,王东阳,王丹,等.木薯块根膨大期韧皮部和木质部比较蛋白组学初步研究[J].热带作物学报,2014,35(3):525-533.
[19]贺庭琪.木薯叶片应答不同光照处理的比较蛋白质组学初步研究[D].海口:海南大学,2013.
[20]王海燕,王斌,王丹,等.等电聚焦强度对木薯叶片蛋白质双向电泳分离结果的影响[J].热带作物学报,2011,32(12):2 367-2 372.
[21]王镜岩.生物化学[M].3版.北京:高等教育出版社,2002.
[22]蒋春云,马秀灵,沈晓艳,等.植物碳酸酐酶的研究进展[J].植物生理学报,2013,49(6):545-550.
[23]郭敏亮,高煜珠.植物的碳酸酐酶[J].植物生理学通讯,1989,3:75-80.
[24]Li K M,Zhu W L,Zeng K,et al.Proteome characterization of cassava(Manihot esculenta Crantz)somatic embryos,plantlets and tuberous roots[J].Proteome Science,2010,8:10.
[25]张杨,卢诚,王文泉.木薯栽培种与野生种叶片光合器官结构和酶学差异[J].中国农学通报,2012,28(25):144-149.
[26]Sato N,Terasawa K,Miyajima K,et al.Organization,developmental dynamics,and evolution of plastid nucleoids[J].International Review of Cytology,2003,232:217-262.
[27]Jianming Fu,Ivana Momcilovic,Vara Prasad P V.Roles of protein synthesis elongation factor EF-Tu in heat tolerance plant[J].Journal of Botany,2012,ID835836,8 pages.
[28]Lee J H,Yoon H S,Yun C H,et al.Molecular characterization of the mitochondrial elongation factor EF-Tu gene in rice(Oryza sativa L.)[J].Plant Cell Reports,2002,21:157-165.
[29]Berg M,Seemüller E.Chromosomal organization and nucleotide sequence of genes coding for the elongation factors G and Tu of the apple prolif-eration phytoplasma[J].Gene,1999,226:103-109.
[30]Yang H P,Zhong H N,Zhou H M.Catalysis of the refolding of urea denatured creatinekinase by peptidyl-prolyl cis-trans isomerase[J].Biochimica et Biophysica Acta,1997,1338(2):147-150.
[31]Távora F J A F,Melo F I O,Pinho J L N de,et al.Yield,crop growth rate and assimilatory characteristics of cassava at the coastal area of Ceará[J].Revista Brasileira de Fisiologia Vegetal,1995,7:81-88.
[2]张鹏,安冬,马秋香,等.木薯分子育种中若干基本科学问题的思考与研究进展[J].中国科学,2013,43(12):1 082-1 089.
[3]贺庭琪,徐兵强,郭安平,等.不同品种木薯叶绿体比较蛋白质组学初步研究[J].热带作物学报,2013,34(6):1 090-1 097.
[4]张根连,范术丽,宋美珍,等.植物蛋白质组学技术研究进展[J].生物技术通报,2011,7:26-30.
[5]Frank G,Pressman E,Ophir R,et al.Transcriptional profiling of maturing tomato(Solanum lycopersicum L.)microspores reveals the involvement of heat shock proteins,ROS scavengers,hormones,and sugars in the heat stress response[J].Journal of Experimental Botany,2009,60(13):3 891-3 908.
[6]Baginsky S,Gruissem W.Chloroplast proteomics:potentials and challenges[J].Journal of Experimental Botany,2004,55(400):1 213-1 220.
[7]Jorrín J,Maldonado A,Castillejo M.Plant proteome analysis:a 2006 update[J].Proteomics,2007,7(16):2 947-2 962.
[8]Schroder W,Kieselbach T.Update on chloroplast proteomics[J].Photosynthesis Research,2003,78:181-193.
[9]Fan P X,Wang X C,Kuang T Y,et al.An efficient method for the extraction of chloroplast proteins compatible for 2-DE and MS analysis[J].Electrophoresis,2009,30(17):3 024-3 033.
[10]Saravanan R S,Rose J C.A critical evaluation of sample extraction techniques for enhanced proteomic analysis of recalcitrant plant tissues[J].Proteomics,2004,4:2 522-2 532.
[11]Wang X C,Li X F,Han H P,et al.A protein extraction method compatible with proteomic analysis for euhalophyte Salicornia europaea L[J].Electrophoresis,2007,28(21):3 976-3 987.
[12]Bradford M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding[J].Analytical Biochemistry,1976,72:248-254.
[13]Laemmli U K.Cleavage of structural proteins during the assembly of the head of bacteriophage T4[J].Nature,1970,227(5 259):680-685.
[14]Wang X C,Wang D Y,Wang D,et al.Systematic comparison of technical details in CBB methods and development of a sensitive GAP stain for comparative proteomic analysis[J].Electrophoresis,2012,33(2):296-306.
[15]王旭初,范鹏祥,李银心.一种适用于质谱分析的简化胶内酶解方法[J].植物生理与分子生物学报,2007,33:449-455.
[16]Shen S H,Jing Y X,Kuang T Y.Proteomics approach to identify wound-response related proteins from rice leaf sheath[J].Proteomics,2003,3(4):527-535.
[17]Arun Goysl,Thomas Betsche,Tolbert N E.Isolation of Intact Chloroplasts from Dunaliella Tertiolecta[J].Plant Physiol,1988,88(2):543-546.
[18]王力敏,王东阳,王丹,等.木薯块根膨大期韧皮部和木质部比较蛋白组学初步研究[J].热带作物学报,2014,35(3):525-533.
[19]贺庭琪.木薯叶片应答不同光照处理的比较蛋白质组学初步研究[D].海口:海南大学,2013.
[20]王海燕,王斌,王丹,等.等电聚焦强度对木薯叶片蛋白质双向电泳分离结果的影响[J].热带作物学报,2011,32(12):2 367-2 372.
[21]王镜岩.生物化学[M].3版.北京:高等教育出版社,2002.
[22]蒋春云,马秀灵,沈晓艳,等.植物碳酸酐酶的研究进展[J].植物生理学报,2013,49(6):545-550.
[23]郭敏亮,高煜珠.植物的碳酸酐酶[J].植物生理学通讯,1989,3:75-80.
[24]Li K M,Zhu W L,Zeng K,et al.Proteome characterization of cassava(Manihot esculenta Crantz)somatic embryos,plantlets and tuberous roots[J].Proteome Science,2010,8:10.
[25]张杨,卢诚,王文泉.木薯栽培种与野生种叶片光合器官结构和酶学差异[J].中国农学通报,2012,28(25):144-149.
[26]Sato N,Terasawa K,Miyajima K,et al.Organization,developmental dynamics,and evolution of plastid nucleoids[J].International Review of Cytology,2003,232:217-262.
[27]Jianming Fu,Ivana Momcilovic,Vara Prasad P V.Roles of protein synthesis elongation factor EF-Tu in heat tolerance plant[J].Journal of Botany,2012,ID835836,8 pages.
[28]Lee J H,Yoon H S,Yun C H,et al.Molecular characterization of the mitochondrial elongation factor EF-Tu gene in rice(Oryza sativa L.)[J].Plant Cell Reports,2002,21:157-165.
[29]Berg M,Seemüller E.Chromosomal organization and nucleotide sequence of genes coding for the elongation factors G and Tu of the apple prolif-eration phytoplasma[J].Gene,1999,226:103-109.
[30]Yang H P,Zhong H N,Zhou H M.Catalysis of the refolding of urea denatured creatinekinase by peptidyl-prolyl cis-trans isomerase[J].Biochimica et Biophysica Acta,1997,1338(2):147-150.
[31]Távora F J A F,Melo F I O,Pinho J L N de,et al.Yield,crop growth rate and assimilatory characteristics of cassava at the coastal area of Ceará[J].Revista Brasileira de Fisiologia Vegetal,1995,7:81-88.