太谷核不育小麦Ms2基因蛋白质的表达差异
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摘要
本试验以鲁麦15为背景的太谷核不育小麦近等基因系(鲁麦15,Ms2鲁麦15和Rht10+Ms2鲁麦15)不同时期的幼穗为材料,从蛋白质组学角度利用双向电泳技术和质谱技术对太谷核不育基因Ms2的差异表达蛋白进行了研究,得出了以下结果:
1、经多次重复建立了适用于小麦幼穗蛋白的双向电泳体系。利用TCA-丙酮提取法提取了供试材料减数分裂期、单核期、二核期的全蛋白质组取得了较好的效果。用PH4-7的24cm线性预制干胶条对所提取的蛋白质进行等电聚焦后,用浓度为12%的聚丙烯酰胺凝胶电泳分离后得到了分离效果较好的电泳图谱。
2、在PH4-7,分子量10-115KD的范围内,在减数分裂期得到约500个蛋白点,单核期、二核期得到600多个蛋白点。大多数等电点在PH4.5-7之间,分子量10-80KD之间居多。各个试验材料的幼穗之间蛋白质表达均存在差异,不同时期的差异蛋白有所不同,即不同时期表达的与育性相关的蛋白不同。
3、选取差异蛋白点,进行胶内酶解后进行MALDI-TOF-MS进行肽指纹图谱分析。利用Mascot软件对SWISSPROT数据库搜索发现三个时期中共有47个差异蛋白质点得分较高,均在50以上。选取了得分值较低(低于50分)的30个差异蛋白点进行了高效液相色谱质谱(HPLC-MS/MS)鉴定,有8个蛋白点获得了较好的肽指纹图谱。
4、鉴定出了与败育相关的蛋白:减数分裂时期鉴定出的弹性蛋白酶抑制剂和50S核糖体蛋白L6,单核期鉴定出的含TPR重复的硫氧还原蛋白TTL1,核酮糖1,5二磷酸羧化酶大亚基和ATP合成酶前体;以及二核期鉴定出的磷酸激酶,醌还原酶2和J结构域蛋白是与育性相关的蛋白。另外,还有一些蛋白可能与育性有关,减数分裂期的查尔酮异构酶,细胞周期蛋白依赖性酶抑制因子1;单核期的F-box蛋白、UDP-葡萄糖4差向异构酶、GTP结合蛋白yptV2;二核期的减数分裂重组蛋白SPO11-2、S-腺苷甲硫氨酸合成酶、ras基因相关蛋白Rab7。
5、对鉴定出的蛋白功能进行分析发现,能量代谢紊乱导致代谢供能不足,信号转导受阻导致细胞抗性减弱,活性氧积累导致细胞中毒,细胞凋亡导致花药发育异常及花粉败育等生理现象很可能导致不育的发生。这些差异蛋白很可能直接或间接地参与了供试材料育性正常发育与败育的某些关键途径。与糖类代谢有关的蛋白,与ABA的合成及分解代谢有关的蛋白可能直接或间接减缓了幼穗的生长发育。
Use Taigu sterile near-isogenic lines of wheat (Lumai 15, Ms2 Lumai 15 and Rht10 + Ms2 Lumai 15) as the materials, and material parts are panicle in different periods. Using of 2-DE technology and mass spectrometry, differentially expressed protein of Ms2 were studied, The main results were as follows:
1. The two-dimensional electrophoresis fitting to the young panicle protein of common wheat has been set up through many repeats.Precipitation protein with TCA-acetone method, tested the total proteins of meiosis, uninucleate and binucleus stage of the three materials and achieved good results. Detected with PH4-7,24cm prefabricated dry linear strips for IEF of the extracted proteins,then separation with the concentration of 12% polyacrylamide gel electrophoresis and gain good results.
2. In the pH4-7,10-115KD,there are about 500 protein spots at meiosis stage, 600 spots at uninucleate stage and binucleus,respectively. Most of the protein in the pH4.5-7(strip alkaline side),10-80KD. The differentiation of the protein expression between the immature ears of experimental lines has been found.
3.The peptite fingerprinting(MALDI-TOF-MS) of 80 protein spots of the three phases were analyzed by matrix-assisted laser desorption time of flight mass spectrometry and used Mascot to search in the swissprot datebase,which identifyed 47 protein spots that had gotten scores above 50.The last 30 protein spots which gain lower scores (less than 50 points) were high performance liquid chromatography-mass spectrometry (HPLC-MS/MS) and 8 protein spots were identified and obtained better peptide fingerprinting.
4. Elastase inhibitor 4 and 50S ribosomal protein L6 were identified on meiosis, TPR repeat-containing thioredoxin TTL1,ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit and mitochondrial ATP synthase precursor were identified at the uninucleate stage; Phosphoglycerate kinase,quinone reductase 2 and J-domain protein were identified at the binucleate stage. In addition, some proteins may be related to fertility: at the meiosis stage, Chalcone--flavonone isomerase, cyclin-dependent inhibitor 1; the F-box protein ORE9, UDP-glucose 4-epimerase , Calcineurin B-like protein 3, GTP-binding protein yptV2 at the stage of binucleus;recombination protein SPO11-2, S-adenosylmethionine synthetase, ras-related protein Rab7 at the binuciate stage.
5. Functional analysis known protein,speculated that male sterility of wheat may be related to energy metabolism disorder,signal transduction blocked,accumulation of reactive oxygen species,et. These physiological phenomena are likely to lead to the occurrence of infertility. These differential proteins may directly or indirectly involved in the certain key ways of the fertility and abortion of the tested materials. Protein with carbohydrate metabolism, and ABA synthesis and catabolism-related proteins may directly or indirectly slow the growth of the young spike of wheat.
1、经多次重复建立了适用于小麦幼穗蛋白的双向电泳体系。利用TCA-丙酮提取法提取了供试材料减数分裂期、单核期、二核期的全蛋白质组取得了较好的效果。用PH4-7的24cm线性预制干胶条对所提取的蛋白质进行等电聚焦后,用浓度为12%的聚丙烯酰胺凝胶电泳分离后得到了分离效果较好的电泳图谱。
2、在PH4-7,分子量10-115KD的范围内,在减数分裂期得到约500个蛋白点,单核期、二核期得到600多个蛋白点。大多数等电点在PH4.5-7之间,分子量10-80KD之间居多。各个试验材料的幼穗之间蛋白质表达均存在差异,不同时期的差异蛋白有所不同,即不同时期表达的与育性相关的蛋白不同。
3、选取差异蛋白点,进行胶内酶解后进行MALDI-TOF-MS进行肽指纹图谱分析。利用Mascot软件对SWISSPROT数据库搜索发现三个时期中共有47个差异蛋白质点得分较高,均在50以上。选取了得分值较低(低于50分)的30个差异蛋白点进行了高效液相色谱质谱(HPLC-MS/MS)鉴定,有8个蛋白点获得了较好的肽指纹图谱。
4、鉴定出了与败育相关的蛋白:减数分裂时期鉴定出的弹性蛋白酶抑制剂和50S核糖体蛋白L6,单核期鉴定出的含TPR重复的硫氧还原蛋白TTL1,核酮糖1,5二磷酸羧化酶大亚基和ATP合成酶前体;以及二核期鉴定出的磷酸激酶,醌还原酶2和J结构域蛋白是与育性相关的蛋白。另外,还有一些蛋白可能与育性有关,减数分裂期的查尔酮异构酶,细胞周期蛋白依赖性酶抑制因子1;单核期的F-box蛋白、UDP-葡萄糖4差向异构酶、GTP结合蛋白yptV2;二核期的减数分裂重组蛋白SPO11-2、S-腺苷甲硫氨酸合成酶、ras基因相关蛋白Rab7。
5、对鉴定出的蛋白功能进行分析发现,能量代谢紊乱导致代谢供能不足,信号转导受阻导致细胞抗性减弱,活性氧积累导致细胞中毒,细胞凋亡导致花药发育异常及花粉败育等生理现象很可能导致不育的发生。这些差异蛋白很可能直接或间接地参与了供试材料育性正常发育与败育的某些关键途径。与糖类代谢有关的蛋白,与ABA的合成及分解代谢有关的蛋白可能直接或间接减缓了幼穗的生长发育。
Use Taigu sterile near-isogenic lines of wheat (Lumai 15, Ms2 Lumai 15 and Rht10 + Ms2 Lumai 15) as the materials, and material parts are panicle in different periods. Using of 2-DE technology and mass spectrometry, differentially expressed protein of Ms2 were studied, The main results were as follows:
1. The two-dimensional electrophoresis fitting to the young panicle protein of common wheat has been set up through many repeats.Precipitation protein with TCA-acetone method, tested the total proteins of meiosis, uninucleate and binucleus stage of the three materials and achieved good results. Detected with PH4-7,24cm prefabricated dry linear strips for IEF of the extracted proteins,then separation with the concentration of 12% polyacrylamide gel electrophoresis and gain good results.
2. In the pH4-7,10-115KD,there are about 500 protein spots at meiosis stage, 600 spots at uninucleate stage and binucleus,respectively. Most of the protein in the pH4.5-7(strip alkaline side),10-80KD. The differentiation of the protein expression between the immature ears of experimental lines has been found.
3.The peptite fingerprinting(MALDI-TOF-MS) of 80 protein spots of the three phases were analyzed by matrix-assisted laser desorption time of flight mass spectrometry and used Mascot to search in the swissprot datebase,which identifyed 47 protein spots that had gotten scores above 50.The last 30 protein spots which gain lower scores (less than 50 points) were high performance liquid chromatography-mass spectrometry (HPLC-MS/MS) and 8 protein spots were identified and obtained better peptide fingerprinting.
4. Elastase inhibitor 4 and 50S ribosomal protein L6 were identified on meiosis, TPR repeat-containing thioredoxin TTL1,ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit and mitochondrial ATP synthase precursor were identified at the uninucleate stage; Phosphoglycerate kinase,quinone reductase 2 and J-domain protein were identified at the binucleate stage. In addition, some proteins may be related to fertility: at the meiosis stage, Chalcone--flavonone isomerase, cyclin-dependent inhibitor 1; the F-box protein ORE9, UDP-glucose 4-epimerase , Calcineurin B-like protein 3, GTP-binding protein yptV2 at the stage of binucleus;recombination protein SPO11-2, S-adenosylmethionine synthetase, ras-related protein Rab7 at the binuciate stage.
5. Functional analysis known protein,speculated that male sterility of wheat may be related to energy metabolism disorder,signal transduction blocked,accumulation of reactive oxygen species,et. These physiological phenomena are likely to lead to the occurrence of infertility. These differential proteins may directly or indirectly involved in the certain key ways of the fertility and abortion of the tested materials. Protein with carbohydrate metabolism, and ABA synthesis and catabolism-related proteins may directly or indirectly slow the growth of the young spike of wheat.
引文
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