苹果属植物树皮组织总蛋白提取及分离方法的建立
? 共同第一作者
收稿日期: 2014-10-27
录用日期: 2015-01-27
网络出版日期: 2015-09-06
基金资助
农业部现代农业产业技术体系建设专项(No;CARS-28)、国家科技支撑计划(No.2013BAD02B01)和国家自然科学基金(No;30900968, No.31201602)
Extraction and Separation Analysis of Total Protein from the Bark of Apple Branches
? These authors contributed equally to this paper
Received date: 2014-10-27
Accepted date: 2015-01-27
Online published: 2015-09-06
为了建立适于苹果属植物树皮组织总蛋白提取的技术方法, 以8年生华月苹果(Malus domestica)树枝条为试材, 通过比较不同提取方法(TCA-丙酮沉淀法(A)、甲醇/醋酸铵沉淀法(B)和改良的Tris-酚抽提方法(C))并优化提取条件, 确立了最适提取及分离方法为改良的Tris-酚抽提方法。在2-DE分离时, 该方法所获得的样品图谱中蛋白点总数为993个, 明显多于TCA-丙酮沉淀(418个)和甲醇醋酸铵沉淀(674个)方法, 并且与其它两种方法相比, 该方法获得的图谱背景更清晰, 蛋白点聚焦效果更好。此外, 经过3个梯度上样量的图谱分离效果比较, 确定了800 μg为本研究中2-DE分析的理想上样量。另外, 为了验证该提取及分离方法的可行性, 进一步对蛋白质表达谱中的部分蛋白点进行了质谱分析, 且这些蛋白点均得到了成功鉴定。该研究通过优化总蛋白提取方法及样品上样量等条件, 获得了理想的双向电泳分离图谱, 为苹果属植物树皮组织材料的蛋白质组学研究奠定了基础。
张彩霞, 田义, 张利义, 肖龙, 丛佩华 . 苹果属植物树皮组织总蛋白提取及分离方法的建立[J]. 植物学报, 2015 , 50(6) : 739 -745 . DOI: 10.11983/CBB14186
We aimed to establish a suitable extraction method for analyzing total protein in the bark of apple branches. The branches of an 8-year-old cultivar (Huayue) were used as material. We compared 3 different extraction methods, including TCA-acetone precipitation (A), methanol/ammonium acetate precipitation (B), and improved Tris-phenol extraction (C). We optimized the extraction procedures and established an ideal method for analyzing the total protein in the bark of apple branches. The improved Tris-phenol extraction method could reveal a good map of 993 protein spots, significantly more than the other two methods: 418 and 674 protein spots were obtained with TCA-acetone precipitation and methanol/ammonium acetate precipitation, respectively. Furthermore, the gel background was clear, and the protein spots were better focused than with the two other methods. After comparing sample loading quantity, we selected 800 μg as the ideal loading quantity for our study. Furthermore, we selected several proteins for MALDI-TOF-TOF/MS analysis and database searching, and the protein spots were finally identified. In this study, we optimized the extraction procedures and sample loading quantity for total protein and finally obtained an ideal 2-D electrophoresis map. Our study may provide a good basis for further proteome analysis of apple branches.
| [1] | 曹尚银, 张秋明, 朱志勇, 郭俊英, 陈玉玲, 薛华柏 (2007). 苹果花芽孕育蛋白质组学初步分析. 中国农业科学 40, 2281-2288. |
| [2] | 丁承强, 马丹, 王绍华, 丁艳锋 (2011). 水稻蛋白质组双向电泳优化流程及方法. 植物学报 46, 67-73. |
| [3] | 谷瑞升, 刘群录, 陈雪梅, 蒋湘宁 (1999). 木本植物蛋白提取和SDS-PAGE分析方法的比较和优化. 植物学通报 16, 171-177. |
| [4] | 刘欣颖, 吕松, 王忆, 王昆, 李天红, 韩振海, 张新忠 (2011). 苹果种质资源对苹果树腐烂病抗性评价. 果树学报 28, 843-848. |
| [5] | 王清, 产祝龙, 秦国政, 田世平 (2009). 果实蛋白质组学研究的实验方法. 植物学报 44, 107-116. |
| [6] | 王一鸣, 花宝光, 王有年, 孟海玲 (2007). 桃果实蛋白质双向电泳影响因素的研究. 园艺学报 34, 1579-1584. |
| [7] | 曾广娟, 李春敏, 张新忠, 田义, 陈东玫, 赵永波, 董文轩 (2008). 苹果实生树阶段转变特异蛋白质的SDS-PAGE分析. 园艺学报 35, 1059-1064. |
| [8] | 张彩霞, 李壮, 张利义, 田义, 康国栋, 陈莹, 丛佩华 (2012). 苹果叶片总蛋白提取及其双向电泳分析. 果树学报 29 |
| [8] | 1)488-492. |
| [9] | 张玉经, 王昆, 王忆, 韩振海, 高源, 许雪峰, 张新忠 (2010). 苹果种质资源果实轮纹病抗性的评价. 园艺学报 37, 539-546. |
| [10] | 朱志勇, 曹尚银, 徐小彪, 郭俊英, 陈玉玲, 薛华柏 (2007). 新红星苹果花芽蛋白质提取及双向电泳的改良方法. 果树学报 24, 549-552. |
| [11] | Buron-Moles G, Torrres R, Amoako-Andoh F, Viñas I, Teixidó N, Usall J, Keulemans W, Davey MW (2014). Analysis of changes in protein abundance after wounding in ‘Golden Delicious’ apples.Postharv Biol Technol 87, 51-60. |
| [12] | Cao X, Gao Y, Wang Y, Li CM, Zhao YB, Han ZH, Zhang XZ (2011). Differential expression and modification of proteins during ontogenesis inMalus domestica. Proteomics 11, 4688-4701. |
| [13] | Carpentier SC, Witers E, Laukens K, Deckers P, Swennen R, Panis B (2005). Preparation of protein extracts from recalcitrant plant tissues: an evaluation of different methods for two-dimensional gel electrophoresis analysis. Proteomics 5, 2497-2507. |
| [14] | Gao KX, Liu XG, Liu YG, Zhang TB, Wang SL (2002). Potential of Trichoderma harzianum and T. atroviride to control Botryosphaeria berengeriana f. sp. piricola, the cause of apple ring rot.J Phytopathol 150, 271-276. |
| [15] | Guarino C, Arena S, De Simona L, D'ambrosio C, Santoro S, Rocco M, Scaloni A, Marra M (2007). Proteomic analysis of the major soluble components in Annurca apple flesh.Mol Nutr Food Res 51, 255-262. |
| [16] | Jiang L, He L, Fountoulakis M (2004). Comparison of protein precipitation methods for sample preparation prior to proteomic analysis .J Chromatogr A 1023, 317-320. |
| [17] | Qin GZ, Wang Q, Liu J, Li BQ, Tian SP (2009). Proteomic analysis of changes in mitochondrial protein expression during fruit senescence. Proteomics 9, 4241-4253. |
| [18] | Rampitsch C, Bykova NV (2012). Proteomics and plant disease: advances in combating a major threat to the global food supply.Proteomics 12, 673-690. |
| [19] | Song J, Braun G, Bevis E, Doncaster K (2006). A simple protocol for protein extraction of recalcitrant fruit tissues suitable for 2-DE and MS analysis.Electrophoresis 27, 3144-3151. |
| [20] | Velasco R, Zharkikh A, Affourtit J, Dhingra A, Cestaro A, Kalyanaraman A, Fontana P, Bhatnagar SK, Troggio M, Pruss D, Salvi S, Pindo M, Baldi P, Castelletti S, Cavaiuolo M, Coppola G, Costa F, Cova V, Dal Ri A, Goremykin V, Komjanc M, Longhi S, Magnago P, Malacarne G, Malnoy M, Micheletti D, Moretto M, Perazzolli M, Si-ammour A, Vezzulli S, Zini E, Eldredge G, Fitzgerald LM, Gutin N, Lanchbury J, Macalma T, Mitchell JT, Reid J, Wardell B, Kodira C, Chen ZT, Desany B, Niazi F, Palmer M, Koepke T, Jiwan D, Schaeffer S, Krishnan V, Wu CJ, Chu VT, King ST, Vick J, Tao QZ, Mraz A, Stormo A, Stormo K, Bogden R, Ederled D, Stella A, Vecchidtti A, Kater MM, Masiero S, Lasserre P, Lespinasse Y, Allan AC, Bus V, Chagné D, Crowhurst RN, Gleave AP, Lavezzo E, Fawcett JA, Proost S, Rouzé P, Sterck L, Toppo S, Lzaazri B, Hellens RP, Durel CE, Gutin A, Bumgarner RE, Gardiner SE, Skolnick M, Egholm M, Van de Peer Y, Salamini F, Viola R (2010). The genome of the domesticated apple (Malus × domestica Borkh.).Nature Genet 42, 833-839. |
| [21] | Wang JY, Ruan SL, WU WH, Xu XF, Wang Y, Han ZH (2010). Proteomics approach to identify differentially expressed proteins induced by iron deficiency in roots ofMalus. Pak J Bot 42, 3055-3064. |
| [22] | Wang XC, Li XF, Deng X, Han HP, Shi WL, Li YX (2007). A protein extraction method compatible with proteomic analysis for the euhalophyteSalicornia europaea. Electrophoresis 28, 3976-3987. |
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