棉花晋A细胞质雄性不育系及其保持系差异蛋白质组分析

doi:10.3864/j.issn.0578-1752.2014.20.001

摘要/Abstract

摘要： 【目的】对棉花晋A细胞质雄性不育系及其同核异质保持系MB177（JB）雄性败育关键时期的花药进行差异蛋白质组研究，为进一步揭示棉花细胞质雄性不育机理奠定基础。【方法】运用双向电泳技术对晋A细胞质雄性不育系及其保持系小孢子发育的造孢细胞时期和小孢子母细胞时期表达的蛋白质进行分离（考马斯亮蓝染色）；采用PDQuest8.0.1软件进行斑点检测，并经统计学分析确定获得差异表达蛋白质；对这些差异蛋白质斑点进行LC-Chip-ESI-QTOF-MS质谱分析，用Mascot软件搜索NCBInr绿色植物，鉴定差异表达蛋白质；对差异表达蛋白质进行GO和KEGG功能分析。【结果】PDQuest8.0.1软件分析表明，晋A不育系和保持系在花药发育的造孢细胞时期和小孢子母细胞时期的花蕾总蛋白质斑点数分别为1 525、1 540和1 554、1 540，这些蛋白质斑点的分子量分布在10—100 kD，等电点分布在3—10。经差异定量研究和统计学分析，共鉴定到15个在晋A不育系与保持系间显著差异表达的蛋白斑点，15个差异蛋白质斑点都得到了质谱特异峰图。对应的蛋白质H（+）-转运ATP合成酶、谷胱甘肽还原酶、线粒体上的ATP酶亚基、核酮糖-1,5-二磷酸羧化酶/加氧酶大亚基和UDP-D葡萄糖脱氢酶在不育系与保持系花药发育2个阶段均差异表达；膜联蛋白、S-甲酸谷胱甘肽水解酶、momilactone A 合成酶类似物、一个具有丙二烯氧化环化酶活性的未命名蛋白质和一个位于线粒体上的保守预测蛋白质仅在不育系和保持系花药发育的造孢细胞时期差异表达；β-羟酰-ACP脱水酶、丙酮酸脱氢酶-α亚基以及与花粉发育相关的一个预测蛋白质仅在不育系和保持系花药发育的小孢子母细胞时期差异表达。这些差异表达蛋白质主要参与小孢子与绒毡层发育过程，它们的下调或上调表达可能造成发育与代谢过程的不协调性，产生不正常的小孢子和绒毡层提前解体，从而导致雄性不育。采用荧光定量PCR分析核酮糖-1,5-二磷酸羧化酶/加氧酶大亚基基因和H（+）转运ATP合成酶基因的转录变化，它们在转录水平与蛋白质表达的趋势一致。【结论】晋A不育系的小孢子败育可能与能量代谢紊乱、茉莉酸合成途径损害、细胞内大量ROS积累、查尔酮合酶活性下降有关。雄性不育的产生是一个复杂的生物学过程，涉及到多个基因的相互作用，构成了一个复杂的调控网络。

关键词: 棉花, 细胞质雄性不育, 蛋白质组学, 双向凝胶电泳

Abstract: 【Objective】This study was conducted to analyze and compare proteomes of cotton flower buds at the microspore abortion stage between a cytoplasmic male sterile (CMS) line JA-CMS and its maintainer line JB.【Method】Two-dimensional gel electrophoresis (2-DE) technique was used to separate the protein spots of JA-CMS and JB flower buds at the microspore abortion stage and the gel was stained with Coomassie Blue R-350 solution. Differentially expressed (DE) protein spots were selected with more than 2-fold changes and P values less than 0.05 by PDQuest (version 8.0.1) image software. LC-Chip/ESI-QTOF-MS analysis was carried out to obtain peptide mass fingerprinting of the DE protein spots. The Mascot software was used to search against the NCBInr database for protein annotation. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analyses were performed to further explore the biological functions of the DE proteins.【Result】The 2-DE maps of JA-CMS at the sporogenous cells stage (SS) and microsporocyte stage (MS) isolated 1 525 and 1 540 protein spots, respectively, while 1 554 and 1 540 protein spots were detected in JB at the same stages. These protein spots were found within Mr 10-100 kD and pI 3-10. Fifteen DE protein spots between JA-CMS and JB were selected after the quantitative and statistical analysis. Among the 15 proteins, H(+)-transporting ATP synthase, glutathione reductase, ATPase subunit 1 (mitochondrion), ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit, and UDP-D-glucose dehydrogenase were differentially expressed at both the SS and MS stages between JA-CMS and JB. Anx1, S-formylglutathione hydrolase, momilactone A synthase-like, an unnamed protein product with allene oxide cyclase activity, and a conserved hypothetical protein located in mitochondria were down-regulated only at the SS stage in JA-CMS; beta-hydroxyacyl-ACP dehydratase, pyruvate dehydrogenase alpha subunit, and a predicted protein related with pollen development were differentially expressed only at the MS stage between the two materials. Functional category analysis indicated that these DE proteins were mainly involved in processes crucial for microspore and tapetum development. Down- and up-regulation of these proteins may disrupt the coordination of developmental and metabolic processes, resulting in abnormal microspores and defective tapetum which leads to male sterility in JA-CMS ultimately. Quantitative RT-PCR was used to validate Rubisco and H(+)-transporting ATP synthase gene expressions at the transcriptional level, which are consistent with the protein amounts detected dy 2-DE.【Conclusion】CMS phenotype of JA-CMS may be associated with energy metabolism disturbance, abnormal jasmonic acid biosynthesis, accumulation of reactive oxygen species (ROS), and decreased activity of chalcone synthase (CHS). CMS is regulated by a complex signal network, multiple genes of different metabolic pathways may affect pollen fertility.

Key words: cotton, cytoplasmic male sterility (CMS), proteomics, 2-DE

杨鹏，韩锦峰，黄晋玲. 棉花晋A细胞质雄性不育系及其保持系差异蛋白质组分析[J]. 中国农业科学, 2014, 47(20): 3929-3940.

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