大豆质核互作雄性不育系NJCMS1A及其保持系的花药差异蛋白质组学研究

Proteomic Study of Anther Differentiation Between Cytoplasmic-Nuclear Male-Sterile Line NJCMS1A and Its Maintainer in Soybean[Glycine max (L) Merr.]

【目的】对大豆质核互作雄性不育系NJCMS1A及其同型保持系NJCMS1B的二胞花粉期花药进行差异蛋白质组学研究,探讨大豆质核互作雄性不育的分子机制。【方法】采用双向凝胶电泳技术对其蛋白质进行分离,凝胶用考马斯亮蓝染色,使用PDQuest软件分析蛋白质图谱,利用基质辅助激光解吸飞行时间质谱(MALDI-TOF-MS)技术对差异表达蛋白进行质谱分析,获得肽质量指纹图谱,用Profound和Mascot软件搜索NCBInr数据库,初步鉴定差异表达蛋白并分析其功能。【结果】在分子量18.4～116.0kD、等电点4～7线性范围内,检测到约212个蛋白点,差异表达蛋白点24个。其中10个在NJCMS1A中出现而在NJCMS1B中缺失,12个在NJCMS1A中缺失而在NJCMS1B中出现,2个表达量在NJCMS1B中比在NJCMS1A中明显增强。鉴定出11个差异表达蛋白,其中7个在NJCMS1A中出现而在NJCMS1B中缺失,4个在NJCMS1A中缺失而在NJCMS1B中出现。【结论】对主要差异表达蛋白如ACC氧化酶、半胱氨酸蛋白酶、V型H+-ATP酶A亚基、MADS盒蛋白、淀粉分枝酶和UDP-葡萄糖焦磷酸化酶等进行功能分析,推测不育系NJCMS1A雄性不育性可能与能量代谢紊乱、细胞程序化死亡(PCD)、乙烯过度合成、淀粉合成受抑制和花器官发育调节基因作用失控等有关。

【Objective】 The present paper was aimed at the differential proteomic study of binucleate anther pollen stage between the cytoplasmic-nuclear male-sterile line NJCMS1A and its maintainer NJCMS1B in soybean. 【Method】 Two-dimensional gel electrophoresis (2-DE) technique was used to separate the protein spots while gels was stained with the Coomassie Blue G-250 .The difference between protein maps of anthers from NJCMS1A and NJCMS1B was analyzed with PDQuest image software. Matrix-assisted laser-adsorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) technique was used to obtain the peptide mass fingerprinting of differentially expressed protein spots. The Profound and Mascot software were used to search the protein database NCBInr to identify those spots interested. 【Result】 About 212 protein spots were detected within Mr 18.4-116.0 kD and pH 4-7. A total of 24 spots out of 212 were found differentially expressed between NJCMS1A and NJCMS1B. Among these, 10 protein spots were present in the protein map of NJCMS1A anther but absent in that of NJCMS1B, 12 present in that of NJCMS1B but absent in that of NJCMS1A, and two up-regulated significantly in that of NJCMS1B in comparison with that ofNJCMS1A. A total of 11 protein spots from 24 were identified, including seven spots present in NJCMS1A anther but absent in NJCMS1B and four spots absent in NJCMS1A but present in NJCMS1B. 【Conclusion】The functions, especially those related to male sterility of the major differentially expressed proteins, including 1-aminocyclopropane-1-carboxylate oxidase (ACC oxidase), cysteine proteinase, vacuolar H+-ATPase A subunit, MADS box protein, starch branching enzyme and UDP-glucose pyrophosphorylase were reviewed and discussed. It was inferred that the male sterility of NJCMS1A might be related to energy metabolism turbulence, programmed cell death (PCD), ethylene excessive synthesis, starch synthesis suffocation and disturbed function of the flower developmental gene.