【作者】 曾维英；

【导师】 杨守萍；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2007， 硕士

【摘要】 质核互作雄性不育性在农作物杂种优势利用中起着重要作用，揭示质核互作雄性不育性的机理具有重要的理论和实际意义，关于植物雄性不育的蛋白质组学研究的报道不多，尤其在大豆上尚未见到相关报道。本文对大豆质核互作雄性不育系NJCMS1A和NJCMS2A与其对应保持系NJCMS1B和NJCMS2B的不同器官的蛋白质组差异进行比较研究，获得如下结果：1．利用蛋白质组技术分析大豆质核互作雄性不育系NJCMS1A和NJCMS2A与其对应保持系NJCMS1B和NJCMS2B的不同器官的蛋白质组差异，结果发现不育系与保持系的花药间差异表达蛋白较多，种子间仅有少量的差异表达蛋白，而叶片间2-DE图谱基本一致，几乎没有差异，说明蛋白表达具有时空性。2．运用MALDI-TOF-MS质谱技术鉴定不育系NJCMS1A与其保持系NJCMS1B的花药中差异表达蛋白，单核小孢子期花药中鉴定出7个差异表达蛋白，二胞花粉期花药中鉴定出16个差异表达蛋白，对主要差异蛋白包括ACC氧化酶、ACC合成酶、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)、乙烯过度产生、淀粉合成受抑制和花器官发育调节基因作用失控等有关。3．运用MALDI-TOF-MS质谱技术分析不育系NJCMS2A与其保持系NJCMS2B的花药中差异表达蛋白，单核小孢子期花药中鉴定出4个差异表达蛋白，二胞花粉期花药中鉴定出14个差异表达蛋白，对主要差异蛋白如热激蛋白22 kD、半胱氨酸蛋白酶、V型H~+-ATP酶A亚基、MADS盒蛋白和淀粉分枝酶等进行功能分析，推测不育系NJCMS2A雄性不育性可能与能量代谢紊乱、细胞程序化死亡(PCD)、淀粉合成受抑制和花器官发育调节基因作用失控等有关。4．分析发现在不育系NJCMS1A和NJCMS2A的单核小孢子期和二胞花粉期花药中均缺失而在保持系NJCMS1B和NJCMS2B中均出现的蛋白有MADS盒蛋白和淀粉分枝酶；在不育系NJCMS1A和NJCMS2A的二胞花粉期花药中均出现而在保持系NJCMS1B和NJCMS2B中均缺失的蛋白有半胱胺酸蛋白酶、V型H~+-ATP酶A亚基、腺苷酸脱氨酶、拟南芥1号染色体基因组编码蛋白、寡尿苷酸结合酶、Cullin、β-香树素合成酶和Hypothetical protein MtrDRAFT_AC146570g8v1。这些蛋白可能对育性的影响比较稳定，建议在以后的研究中，选择上述蛋白进行基因克隆，以进一步确定其功能。更多还原

【Abstract】 Cytoplasmic-nuclear male sterility is a mojor tool in the hybrid seed production for utilization of heterosis in crops. The knowledge of the mechanism of the cytoplasmic-nuclear male-sterility is essential to hybrid seed production. Few studies have been done on the proteomics of the male sterility in crops, especially no relevant paper has been reported in soybean so far. The soybean cytoplasmic-nuclear male-sterile lines NJCMS1A and NJCMS2A were developed through consecutive backcross procedures in the National Center for Soybean Improvement, Nanjing Agricultural University. The present paper was aimed at the differential proteomic studies of the different organs between NJCMS1A, NJCMS2A and their maintainers NJCMSIB, NJCMS2B respectively. The results were as follows:1.The proteomic approach was used to detect the differentially expressed proteins of the different organs between the male-sterile lines NJCMS 1 A, NJCMS2A and their maintainers NJCMS IB, NJCMS2B respectively. The results showed that the differences were investigated on the 2-DE maps of anthers and seeds, but not found on those of leaves between NJCMS1A, NJCMS2A and NJCMS1B, NJCMS2B respectively. The above results showed that the expression of the proteins related to the male sterility were spacially and periodly.2.The matrix-assisted laser-adsorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) technique was used to obtain the peptide mass fingerprinting of the differentially expressed proteins in anthers between NJCMS1A and NJCMS1B. In the anthers at uninucleate microspore stage, 7 differentially expressed protein spots were identified. In the anthers at binucleate pollen stage, 16 differentially expressed protein spots were identified. According to the literature, the functions, especially those related to the male sterility of the major differentially expressed proteins, including 1-aminocyclopropane-1-carboxylate oxidase (ACC oxidase), ACC synthase 2, cysteine proteinase, vacuolar H~+-ATPase A subunit, MADS box protein, starch branching enzyme, and UDP-glucose pyrophosphorylase etc. were reviewed and discussed. It was inferred that the male sterility of NJCMS1A might be related to energy metabolism turbulence, the programmed cell death (PCD), ethylene excessive synthesis, starch synthesis suffocation and the abnormal function of the flower developmental gene.3.The matrix-assisted laser-adsorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) technique was used to obtain the peptide mass fingerprinting of the differentially expressed proteins in anthers between NJCMS2A and NJCMS2B. In the anthers at uninucleate microspore stage, 4 differentially expressed protein spots were identified. In the anthers at binucleate pollen stage, 14 differentially expressed protein spots were identified. According to the literature, the functions, especially those related to the male sterility of the major differentially expressed proteins, including Heat shock 22 kDa protein, cysteine proteinase, vacuolar H+-ATPase A subunit, MADS box protein, and starch branching enzyme were reviewed and discussed. It was inferred that the male sterility of NJCMS2A might be related to energy metabolism turbulence, the programmed cell death (PCD), starch synthesis suffocation and the abnormal function of the flower developmental gene.4.To synthesize the results of NJCMS1A and NJCMS2A, it was found that the MADS box protein and starch branching enzyme didn’t appear in the anthers at both uninucleate microspore and binucleate pollen stages of NJCMS1A and NJCMS2A, but appeared in those of NJCMS1B and NJCMS2B; cysteine proteinase, vacuolar H~+-ATPase A subunit, adenosine/AMP deaminase, AIG1-like protein, oligouridylate binding protein, cullin, beta-amyrin synthase and hypothetical protein MtrDRAFT_AC146570g8vl appeared in the anthers at binucleate pollen stages of NJCMS1A and NJCMS2A, but didn’t appear in those of NJCMS1B and NJCMS2B. These proteins might be related to the male sterility of NJCMS1A and NJCMS2A stably. It was suggested to clone the genes of the above proteins and study their functions, especially those related to the male sterility.更多还原