巴西橡胶树橡胶粒子蛋白质的16-BAC/SDS-PAGE双向电泳及质谱分析

doi:10.3864/j.issn.0578-1752.2012.11.023

摘要/Abstract

摘要： 【目的】分析巴西橡胶树（Hevea Brasiliensis Müll.Arg.）橡胶粒子的蛋白质构成，探索未知的橡胶粒子蛋白质。【方法】采用16-BAC/SDS PAGE双向电泳分离橡胶粒子蛋白质，采用MALDI/TOF-TOF质谱技术进行蛋白质鉴定；此外，利用磺基异硫氰酸苯酯（SPITC）化学辅助质谱测序法进行肽段的从头测序，获得未知蛋白质肽段的氨基酸序列。通过搜索橡胶树胶乳转录组测序获得的EST数据库，获取已知部分氨基酸序列蛋白质的全序列，或通过部分肽段获取为未知蛋白质编码的EST序列。【结果】获得了橡胶粒子蛋白质的16-BAC/SDS-PAGE双向电泳图谱，鉴定了17个橡胶粒子蛋白质；获得1个小橡胶粒子蛋白（small rubber particle protein，SRPP）亚家族成员SRPP(gi|37622210)的完整氨基酸序列以及1个橡胶延伸因子（rubber elongation factor，REF）亚家族新成员(hbREF2)的C端87个氨基酸残基序列，此蛋白质的氨基酸序列与已知的REF(gi|38122474)具有高度的相似性。【结论】采用16-BAC/SDS-PAGE双向电泳有效地分离了橡胶粒子蛋白质，展现了橡胶粒子蛋白质的相对含量，研究结果表明构成橡胶粒子的主要蛋白质组分是REF和SRPP亚家族蛋白质。橡胶粒子的蛋白质组分的全面鉴定，将有助于揭示橡胶粒子的生物学功能。

关键词: 巴西橡胶树, 橡胶粒子, 蛋白质, 16-BAC/SDS-PAGE双向电泳, 质谱分析

Abstract: 【Objective】 The objective of the study is to investigate the protein composition and to identify the unknown proteins of the rubber particles in the latex of a rubber tree (Hevea brasiliensis Müll. Arg.).【Method】The rubber particle proteins were separated using two dimensional 16-BAC/SDS-PAGE. The protein spots were cut from the 2-DE gel, subjected to trypsin digestion, and then analyzed using MALDI-TOF-TOF or mass spectrometry de novo sequencing after 4-sulfophenyl isothiocyanate (SPITC) derivatization of tryptic peptides. 【Result】 A total of 17 rubber particle proteins were identified through a MALDI/ TOF-TOF analysis, among which a new member of the rubber elongation factor (REF) subfamily, hbREF2, was identified using a method of SPITC based de novo amino acid sequencing, and its partial 3’ terminal sequence with 87 amino acid residues was obtained. The amino acid sequence of hbREF2 had a high degree of similarity with that of a well-known REF member (gi|38122474). In addition, the complete amino acid sequence of one member in the small rubber particle protein (SRPP) subfamily, SRPP (gi|37622210), was obtained through searching the latex transcriptome ESTs database with peptide sequences.【Conclusion】Rubber particle proteins were effectively separated by a 16-BAC/SDS-PAGE method and characterized by mass spectrometry. The data showed the relative abundances of rubber particle proteins and demonstrated that the major protein components of rubber particles are REF and SRPP isoforms. The global identification of the rubber particle proteins is essential to elucidate the biological functions of the rubber particles in the latex of rubber trees.

Key words: Hevea brasiliensis, rubber particle, protein, 16-BAC/SDS-PAGE two dimensional electrophoresis, mass spectrometry

代龙军, 项秋兰, 黎瑜, 聂智毅, 康桂娟, 段翠芳, 曾日中. 巴西橡胶树橡胶粒子蛋白质的16-BAC/SDS-PAGE双向电泳及质谱分析[J]. 中国农业科学, 2012, 45(11): 2328-2338.

DAI Long-Jun, XIANG Qiu-Lan, LI Yu, NIE Zhi-Yi, KANG Gui-Juan, DUAN Cui-Fang, ZENG Ri-Zhong. Rubber Particle Protein Analysis of Hevea brasiliensis by Two Dimensional 16-BAC/SDS-PAGE and Mass Spectrometry[J]. Scientia Agricultura Sinica, 2012, 45(11): 2328-2338.

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