摘要
蛋白质是生命功能的执行体,蛋白质组学是研究细胞、组织或生物体中蛋白质组成及其变化规律的科学,目前蛋白质组学在植物上的研究已经陆续展开,但在棉花上还没有被广泛应用。双向电泳作为蛋白质组研究的三大核心技术之一,是目前常用的唯一一种能够连续在一块胶上分离数千种蛋白质的方法,广泛应用于生物学研究的各个方面。
利用双向电泳技术(two-dimensional electrophoresis,2-DE)对植物发育过程中基因表达进行分析,能够从蛋白质水平揭示植物发育的内在机制。在植物雄性不育分子机制的研究中,全蛋白双向电泳技术正得到越来越多的应用。但是迄今为止,关于棉花雄性不育蛋白质组学的研究,国内外未见报道。利用蛋白质组学技术分析棉花花药总蛋白,旨在找到花粉发育过程中与败育相关的蛋白质,为更进一步研究棉花及其他植物败育发生的机理提供依据。
本实验陆地棉花药为材料,通过对蛋白质提取方法、等电聚焦方法、样品上样量等因素进行比较和优化,以期提高双向电泳的分辨率和重复性,试图建立一套适用于棉花花药蛋白质组学分析的双向电泳方法,为使蛋白质组学更好的应用于棉花提供一些借鉴。将棉花花药用液氮研磨,然后用TCA／丙酮沉淀法和苯酚抽提结合甲醇／醋酸铵沉淀法提取蛋白。采取载体两性电解质PH梯度等电聚焦/SDS-PAGE和固相PH梯度等电聚焦/SDS-PAGE双向凝胶电泳,对陆地棉花药总蛋白质进行了分离。结果表明,苯酚抽提结合甲醇／醋酸铵沉淀的方法较适合棉花花药蛋白质的提取,采用载体两性电解质PH梯度聚焦方法较PH梯度等电聚焦方法的效果好,凝胶用硝酸银染色,上样量为500ug时,得到的电泳图谱分辨率高、重复性好,经ImageMaster2D platinum 5.5软件分析后,大约可识别1300-1400个蛋白质点。
采用载体两性电解质pH梯度-SDS-PAGE双向电泳对陆地棉核雄性不育株和可育株双核期花药总蛋白质进行了分离,通过银染显色,获得了分辨率和重复性较好的双向电泳图谱。ImageMaster2D platinum 5.5软件可识别约1300个蛋白质点,其中差异表达的蛋白质数为79个.将其中61个差异点采用基质辅助激光解析电离飞行时间质谱(matrix a ssisted laser de sorption/ionizaton time of flight mass spectrometry, MALDI-TOF-MS)进行了肽指纹图谱分析,通过Mascot软件利用MSDB数据库进行检索,得到了部分差异蛋白的相关信息,并对这些差异蛋白的表达情况、功能等做了比较分析,以期为探讨棉花核雄性不育过程中的基因表达调控机制奠定基础。
质谱分析鉴定出来的蛋白质按功能主要有以下几类：与信号转导相关的蛋白、与基因调控相关的蛋白、与糖代谢及能量代谢有关的酶、与光合作用有关的酶等。推测这些在陆地棉核雄性不育花药中出现的表达量上调或下调的蛋白质或酶类,在棉花败育过程中具有重要的生物学功能。
The functions of life are executed by proteins, proteomics is the study of cells, tissues or organisms in the protein composition and changes in the laws of science. Currently proteomics research are widely used in plants, but in cotton has not been widely used. As one of the three core technologies about proteome research, two-dimensional gel electrophoresis is the only one commonly-used method at present that can continuously separates thousands of proteins in a piece of gel, widely used in all aspects of biological research.
Using two-dimensional gel electrophoresis to analyze the gene expression on the plant development process can reveal the intrinsic mechanisms of plants development in protein level. In research of the molecular mechanism of plant male sterility, the whole protein two-dimensional gel electrophoresis is being used more and more widely. But so far, the study of male-sterile proteomics in cotton has not been reported at home and abroad. Using proteomics to analyze the total proteins of cotton buds, aimed to finding the abortion-related proteins in the process of pollen development, provided the basis for the further studies on the mechanism of abortion occurrence of cotton and the other plants.
This study has established two-dimensional electrophoresis for anther proteomics of upland cotton. We used upland cotton anther as the material, compared and optimized the key steps, such as sample preparation、the volume of the sample, and so on, hoped that improve the resolution and repeatability. We tried to establish a 2-DE method that can be applicable in the cotton proteome analysis.
The materials were frozen in liquid nitrogen immediately and grinded in a mortar, then the proteins were extracted by TCA/acetone precipitation and phenol extraction-methanol/ammonium acetate precipitation. Then proteins were separated by two-dimensional gel electrophoresis. It is expected that phenol extraction-methanol/ammonium acetate precipitation method could be one of the options for protein extraction from cotton anthers, and the ISO-DALT-IEF is better than IPG-DALT-IEF. The high-resolution and well-reproducible patterns of proteins were observed when the silver staining is taken and 500ug protein sample were added in electrophoresis. The 2-DE maps are analyzed using ImageMaster2D platinum 5.5 software, and distinguished approximate 1300-1400 spots.
The proteins of genic male sterility (GMS) upland cotton anther of the sterile line and fertile line were separated by two-dimensional electrophoresis. The silver-stained proteins spots were analyzed using ImageMaster2D platinum 5.5, there were about 1300 detectable spots on each 2DE gel, and about 79 spots were differential expressed. With direct MALDI-TOF mass spectrometry analysis and protein database searching,58 protein spots out of 79 were identified.
Among those proteins, there were putative retinoblastoma binding protein, ibulose-1,5-bisphosphate carboxylase/oxygenase activase 2, ATP-dependent RNA helicase eIF4A-13, glyceraldehyde 3-phosphate dehydrogenase, NADH dehydrogenase subunit 1, gibberellin 20-oxidase, gibberellin 3-hydroxylase 1, cytosolic ascorbate peroxidase 1, polygalacturonase, ketoacyl-CoA synthase, calmodulin-binding heat shock protein, and so on. They were closely associated with signal transduction, Gene regulation, carbohydrate and energy metabolism, Photosynthesis, and so on, all of which are cell activities that are essential to pollen development. Some of the identified proteins were deeply discussed on the relationship to GMS. This study gave new insights into the mechanism of GMS in cotton and demonstrated the power of the proteomic approach in plant biology studies.
引文
1. Kaul, MLH. Male sterility in higher plants[M]. Berlin Heidelberg:Springer Verlag,1988.
2.李泽福,夏加发,唐光勇.植物雄性不育类型及其遗传机制的研究进展[J].安徽农业科学,2000,28(6)：742-746.
3.刘忠松,官春云,陈社员.植物雄性不育机理的研究及应用[M].中国农业出版社,北京：2001,3-5.
4.徐秉芳.核编码的育性恢复基因[J].植物生理学通讯,2000,36(5)：573-580.
5.王学德,张天真,潘家驹.棉花细胞质雄性不育系育性恢复的遗传基础Ⅰ.恢复基因及其遗传效应[J].中国农业科学,1996,29(5)：32-34.
6.王学德,潘家驹.我国棉花细胞质雄性不育系育性恢复的遗传基础Ⅱ.恢复基因与育性增强基因间的互作效应[J].遗传学报,1997,24(3)：271-277.
7.王学德,张天真,潘家驹.细胞质雄性不育陆地棉的细胞效应[J].作物学报,1997,23(4)：393-399.
8.张小全,王学德.细胞质雄性不育陆地棉与海岛棉间杂种优势初步研究[J].棉花学报,2005,17(2)：79-83.
9.贾占昌.棉花雄性不育系104-7A的选育与三系配套[J].中国棉花,1990,17(6)：11.
10.俞志华,王学德.棉花细胞质雄性不育的研究及改良[J].棉花学报,1999,11(5)：268-274
11.黄晋玲,杨鹏,李炳林,等.棉花晋A细胞质雄性不育系小孢子发生的显微和超微结构观察[J].棉花学报,2001,13(5)：259-263.
12.舒克孝.国外棉花雄性不育的研究进展[J].国外农业科技,1981,(3)：1-6.
13.吕有军,付亮,王彩霞,等.国内棉花雄性不育性研究现状、问题、对策[J].种子,2005,24(1)：44-49
14.王斌.棉花核雄性不育种质洞A不育系的遗传鉴定及利用研究[J].作物品种资源,1997,(2)：29-30.
15.张相琼,张东铭,周宏俊,等.棉花光A不育系的遗传鉴定及利用研究[J].西南农业大学学报,1999,21(2)：144-148.
16.何团结,阚画春,路曦结,等.利用核不育系杂交制种的特点与技术[J].中国棉花,2003,30(9)：34-35.
17.邢朝柱,靖深蓉,郭立平,等.转Bt基因抗虫棉双隐性核不育系-中抗A[J].中国棉花,1999,26(6)：27.
18.宇文璞,宇文钢,乔志卫,等.棉花不育系对温度反应研究初报[J].中国棉花,1990,17(2)：19-20.
19.邵圣才，刘辉.棉花温敏雄性不育两用系研究与利用[J].中国棉花,2000,27(12)：18-20.
20.余筱南,陈金湘,李瑞莲,等.棉花温敏雄性不育系的选育与应用研究简报[J].棉花学报,2003,15(6)：380-381.
21.张天真,靖深蓉.杂种棉选育的理论与实践[M].科学出版社,1998.
22.刘少卿,余筱南.棉花雄性不育系的研究进展和展望[J].作物研究,2006,(5)：435-438.
23.汤泽生,余少华,杜素琼,等.棉花“洞庭一号”姊妹系小孢子发育的细胞形态学研究[J].西华师范大学学报：自然科学版,1980,(1)：38-45.
24.李悦有,王学德,徐亚浓.棕色棉细胞质雄性不育花药的细胞学观察[J].浙江大学学报(农业与生命科学版),2002,28(1)：11-15.
25.黄晋玲,杨鹏,李炳林,等.棉花晋A细胞质雄性不育系小孢子发生的显微和超微结构观察[J].棉花学报,2001,13(5)：259-263.
26.刘金兰,聂以春,黄观武,等.棉花“洞A”型核雄性不育材料花粉发育的细胞形态学观察[J].棉花学报,1994,6(2)：70-73.
27.侯磊,肖月华,李先碧,等.棉花晋A雄性不育系花药发育的mRNA差别显示[J].遗传学报,2002,29(4)：359-363.
28.王学德,张天真,潘家驹.细胞质雄性不育棉花小孢子发生的细胞学观察和线粒体DNA和RAPD分析[J].中国农业科学,1998,31(2)：70-75.
29.胡瑞娟,何纪荣,何奕,等.棉花“洞A”雄性不育小孢子败育与蛋白质代谢关系的研究[J].西华师范大学学报,1985,(1)：43-50.
30.宋宪亮,孙学振,王洪刚,等.棉花洞A型核雄性不育系花药败育过程中的生化变化[J].西北植物学报,2004,24(2)：243-247.
31.宋宪亮,孙学振，刘英欣.棉花ms5ms6和雄性不育花药中碳水化合物和游离氨基酸的变化[J].棉花学报,2001,13(6)：334-336.
32.宋宪亮,孙学振,王明林,等.陆地棉双隐性核不育系(ms5ms6)花药发育过程中POD活性和内源激素动态变化初探[J].中国农业科学,2003,36(7)：861-863.
33.王学德.细胞质雄性不育棉花线粒体蛋白质和DNA的分析[J].作物学报,2000,26(1)：35-39.
34.黄晋玲,安泽伟,攀冬丽.棉花晋A细胞质雄性不育系及其保持系的同工酶分析[J].山西农业大学学报,2001,2(3)：233-236.
35.李成奇,石跃进,潘转霞，等.棉花晋A及其保持系酯酶过氧化物酶PAGE电泳分析[J].华北农学报,2004,19(4)：11-13.
36.马小定,邢朝柱.棉花雄性不育研究和应用进展[J].棉花学报,2006,18(5)：309-314
37.李继耕.细胞质雄性不育性的分子机理[J].遗传,1992,14(6)：31-36.
38. Leving CS. The texas cytoplasm of maize:cytoplasmic male sterility and disease susceptibility. Science,1990,250(4983):942-927.
39. Kadowaki K, Suzuki T, Kazama S. A chimeric genecontaining the 5'portion of atp6 is associated with cytoplasmic male sterilityof rice [J]. Mol Gen Genet,1990,224(1):10-16.
40.李继耕.叶绿体遗传与细胞质雄性不育性[J].中国农业科学,1983,1：49-52.
41. Aarts MG, Hodge R, Kalantidis K, et al. The Arabidopsis MALE STERILITY 2 protein shares similarity with reductases in elongation condensation complexes[J]. Plant Journal,1997,12(3): 615-623.
42.刘海河.西瓜17AB核雄性不育两用系的不育机理研究[D].南京农业大学,南京：2005.
43.高俊平,韩广津,宫永超,等.棉花雄性不育的分子生物学研究进展[J].大田农艺,2008,(2)：116-117.
44.黄晋玲.棉花晋A细胞质雄性不育系的遗传研究[D].山西农业大学,太原：2003.
45. Chen K, Meyer VG. Mutation in chloroplast DNA coding for the large subunit of fraction 1 protein correlated with male sterility in cotton[J]. The Journal of Heredity,1979,70(6):431-433.
46. Galau GA, Wilikins TA. Alloplasmic male sterility in AD alloteraploid Goossypium hirsutum upon replacement of its residents A Cytoplasm with that of D Species G-harknessii[J]. Theor Appl Genet, 1989,78:23-30.
47.侯磊,肖月华,李先碧,等.棉花洞A雄性不育系花药发育的mRNA差别显示[J].遗传学报,2002,29(4)：359-363.
48. Humphery SI, Cordwell SJ, Blackstock WP. Complimentarily and limitations with respect to the RNA and DNA worlds[J].Electrophoresis,1997,(18):1217-1242.
49. Wilkina MR, Senchez JC, Williams KL, et al. Why all proteins expressed by genome should be identified and how to do it[J]. Biotechnology and Genetic Engineering Reviews,1995, (13):19-45.
50. O'arrell PH. High resolution two-dimensional electrophoresis of proteins[J]. The Journal of Biological Chemistry,1975,250(10):4007-4021.
51. Patterson SD, Aebersold RH. Proteomics:the first decade and beyond[J]. Nat Genet,2003,33: 311-323.
52. Schmid MB. Structral proteomies.the potential of high-throughput structure determination[J]. Trends Microbiol,2002,10(10Suppl):527-531.
53. Aggarwal K, lee KH. Functional genomics and proteomics as a foundation for systems biology[J]. Briefings in Functional Genomics and Proteomics,2003,2(3):175-184.
54.皇甫海燕,官春云,郭宝顺,等.蛋白质组学及植物蛋白质组学研究进展[J].作物研究,2006,(5)：577-581.
55.罗小敏,崔研,陈彤,等.植物蛋白质组学面临的挑战和前景[J].生物技术通报,2004,(4)：14-18.
56. Park OK. Proteomic studies in plants[J]. Journal of Biochemistry and Mo lecular Biology,2004, 37(1):133-138.
57.梁宇,荆玉祥,沈世华.植物蛋白质组学研究进展[J].植物生态学报,2004,28(1)：114-125.
58.范海延,陈捷,曲波,等.蛋白质组学及其在植物科学研究中的应用[J].生物学通报,2006,41(1)：6-8.
59. Lindermayr C, Saalbach G, Dun IJ. Proteomic identification of nitro sylated proteins in AraM dopsis[J]. Plant Physiology,2005,137(3):921-930.
60. Bestel-Corre G, Dumas-Gaudot E, Poinsot V, et al. Proteome analysis and identification of symbiosis-related proteins from Medicago truncatula Gaertn by two-dimensional electrophoresis and mass spectrometry[J]. Electrophoresis,2002,23(1):122-137.
61. Salekdeh GH, Siopongco J, Wade LJ, et al. Proteomic analysis of rice leaves during drought stress and recovery [J]. Proteomics,2002,2 (9):1131-1145.
62. Cui SX, Huang F, Wang J, et al. A proteomic analysis of cold stress responses in rice seedlings[J]. Proteomics,2005,5(12):3162-3172.
63. Picard P, Bourgoin-Greneche M, Zivy M. Potential of two-dimensional electrophoresis in routine identification of closely related durum wheat lines[J]. Electrophoresis,1997,18(1):174-181.
64. Barreneche T, Bahrman N, Kremer A. Two-dimensional gel electrophoresis confirms the loe level of genetic differentiation between Quercus robur L. and Quercus petraea (Matt) Liebl[J]. Forest Genet, 1996,3(1):89-92.
65. David JL, Zivy M, Cardin ML, et al. Protein evolution in dynamically managed populations of wheat:adaptive responses to macro-environmental conditions[J]. Theoretical and Applied Genetics, 1997,95(5-6):932-941.
66. Herbik A, Giritch A, Horstmann C, et al. Iron and copper-nutrition dependent changes in protein expression in a tomato wild type and the nicotianamine-free mutant chloronerva[J]. Plant Physiology, 1996,111(2):533-540.
67. Santoni V, Bellini C, Caboche M. Use of two- dimensional protein-pattern analysis for the characterization of Arabidopsis thaliana mutants[J]. Planta,1994,192(4):557-566.
68. Komatsu S, Muhammad A, Rakwal R. Seperation and characterization of proteins from green and etiolated shoots of rice (Oryza sativa L.):towards a rice proteome[J]. Electrophoresis,1999,20(3): 630-636.
69. Raharjo TJ, Widjaja I, Roytrakul S, et al. Comparative proteomics of Cannabis sativa plant tissues[J]. Journal of Biomolecular Techniques,2004,15(2):97-106.
70. Fukao Y, Hayashi M, Nishimura M. Proteomic analysis of leaf peroxisomes in greening cotyledons of Arabidopsis thaliana[J]. Plant Cell Physiol,2002,43(7):689-696.
71. Hajduch M, Gapathy A, Stein J W, et al. A systematic proteomic study of seed filling in soybean. Eestablishment of high-resolution two-dimensional reference maps, expression profiles, and an interactive proteome database[J]. Plant Physiol,2005,137(4):1397-1419.
72.薛妙男,杨继华.沙田柚自交、异交花柱蛋白质的双向电泳[J].广西师范大学学报(自然科学版),2000,18(3)：83-85.
73. Bouchez D, Hofte H. Functional genomics in plants[J]. Plant Physiol,1998,118(3):725-732.
74. K Jan van Wijk. Proteomics of the chloroplast:experimentation and prediction[J]. Trends in Plant Science,2000,5(10):420-425.
75. Peltier JB, Friso G Proteomics of the chloroplast:systematic identification and targeting analysis of lumenal and peripheral thylakoid proteins[J]. Plant Cell,2000,12(3):319-341.
76. Millar AH, Sweetlove LJ, Giege P, et al. Analysis of the Arabidopsis mitochondrial proteome[J]. Plant Physiol,2001,127(4):1711-1727.
77. Bae MS, Cho EJ, Choi EY, et al. Analysis of the Arabidopsis nuclear proteome and its response to cold stress[J]. Plant J,2003,36(5):652-663.
78. Joshua LH, Katharine AH, James WA. Harvey Millar. Towards an analysis of the rice mitochondrial proteome[J]. Plant Physiology,2003,132(1):230-242.
79.阮松林,马华升,等.植物蛋白质组学研究进展Ⅱ.蛋白质组技术在植物生物学研究中的应用[J].遗传,2006,28(12)：1633-1648.
80. Tanaka N, Fujita M, Handa H, et al. Proteomics of the rice cell:systematic identification of the protein populations in subcellular compartments[J]. Molecular Genetics and Genomics,2004,271 (5):566-576.
81.梁宇,荆玉祥,沈世华.植物蛋白质组学研究进展[J].植物生态学报,2004,28(1)：114-125.
82.季之娟,薛庆中.植物蛋白质组学研究进展[J].生命科学,2004,16(4)：241-246.
83. Thiellement H, Bahrman N, Damerval C, et al. Proteomics for genetic and physiological studies in plants[J]. Electrophoresis,1999,20(10):2013-2026.
84. Agrawal GK, Yonekura M, Iwahashi Y, et al. System, trends and perspectives of proteomics in dicot plants. Part Ⅲ:Unraveling the proteomes influenced by the environment, and at the levels of function and genetic relationships[J]. Journal of Chromatogr Analytical Technologies in the Biomedical and Life Sciences,2005,815(1-2):137-145.
85. Weiss W, Huber G, Engel KH, et al. Identification and characterization of wheat grain albam in globulifin allergens[J]. Electrophoresis,1997,18 (5):826-833.
86. Imin N, Nizamidin M, Daniher D, et al.Proteomic analysis of somat ic embryogenesis in Medicago truncatua. Explant cultures grown under 6-benzylam inopurine and 1-naphthaleneacetic acid treatments[J]. Plant Physiology,2005,137(4):1250-1260.
87. Costa P, Pionneau C, Bauw G, et al. Separation and characterization of needle and xylem maritimepine proteins[J].Electrophoresis,1999,20(4-5):1098-1108.
88. Barreneche T, Bah rman N, Kremer A. Two-dimensional gel electrophoresis confirms the low level of genetic differentiation between Quercus robur L. and Quercus petraea (Matt) Liebl[J]. Forest Genetics,1996,3 (1):89-92.
89.陈伟,黄春梅,吕柳新.顽拗植物荔枝蛋白质双向电泳的改良方法[J].福建农业大学学报,2001,30(1)：123-126.
90.马凯,高述民,胡青,等.文冠果雄蕊发育的解剖学及雄性不育蛋白的研究[J].北京林业大学学报,2004,26(5)：40-43.
91. Agrawal GK, Yonekura M, Iwahashi Y, et al. System, trends and perspectives of proteomics in dicot plants I. Technologies in proteome establishment[J]. Journal of Chromatography B, Analytical Technologies in the B iomedical and Life Sciences,2005,815 (1-2):109-123.
92.孟慧,段翠芳,曾日中.植物蛋白质组学研究概况[J].热带农业科学,2006,26(2)：60-64.
93. Laver HK, Reynolds SJ, Moneger F, Leaver CJ. Mitochondrial genome organization and expression associated with cytoplasmic male sterility in sunflower[J]. The Plant Journal,1991,1(2):185-193.
94. Horn R, Horstmeyer A, Hahnen J, et al. The CMS-associated 16kD protein encoded by orfH522 in PET1 cytoplasm is also present in other male-sterile cytoplasms of sunflower[J]. Plant Mol.Biol, 1996,30(3):523-538.
95. Song J, Hedgcoth C. A chimeric gene(orf256) is expressed as protein only in cytoplasmic male sterile lines of wheat[J]. Plant Mol.Biol,1994,26(1):535-539.
96. He S, Abad AR, Gelvin SB, et al. A cytoplasmic male sterility-associated mitochondrial protein causes pollen disruption in transgenic tobacco[J]. Proc Natl Acad Sci USA,1996,93(21): 11763-11768.
97.司智海,刘植义.普通小麦T型细胞质雄性不育系及其保持系线粒体多肽的电泳比较研究[J]遗传学报,1991,18(1)：44-50.
98.曾维英,杨守萍,盖钧镒,等.大豆质核互作雄性不育系NJCMS1A和其保持系的不同器官蛋白质组比较研究[J].大豆科学,2008,(1)：8-14.
99.邵锦震,顾勇.水稻剑叶蛋白质的双向电泳分析[J].湖北师范学院学报(自然科学版),2006,26(1)：1-5.
100.魏磊,丁毅,胡耀军,等.紫稻细胞质雄性不育系叶片全蛋白双向电泳分析[J].遗传学报,2002,29(8)：696-699.
101.朱宏,王继华,王同昌.小麦T型细胞质雄性不育系与可育系叶片蛋白质双向电泳分析[J].植物研究,2004,24(3)：98-101.
102.门淑珍,刘博林.小麦显性雄性核不育材料后代不育株与可育株不同器官的蛋白质比较研究
[J].作物学报,2001,27(1)：117-122.
103.藤晓月,陈雪晖.小麦T型细胞质雄性不育系和保持系蛋白质的比较研究[J].作物学报,1996,22(3)：264-270.
104.傅鸿仪.高粱线粒体基因组的翻译产物与细胞质雄性不育性[J].遗传学报,1983,10(6)：471-476.
105. Imin N, Kerim T, Rolfe BG, et al. Effect of early cold stress on the maturation of rice anthers[J]. Proteomics,2004,4(7):1873-1882.
106.文李，刘盖,张再君,等.红莲型水稻细胞质雄性不育花药蛋白质组学初步分析[J].遗传,2006,28(3)：311-316.
107.谢锦云,李小兰,陈平,等.温敏核不育水稻花药蛋白质组初步分析[J].中国生物化学与分子生物学报,2003,19(2)：215-221.
108.曾维英,杨守萍,盖钧镒,等.大豆质核互作雄性不育系NJCMSlA及其保持系的花药差异蛋白质组学研究[J].中国农业科学,2007,40(12)：2679-2687.
109.曾维英,杨守萍,喻德跃,等.大豆质核互作雄性不育系NJCMS2A及其保持系的花药蛋白质组比较研究[J].作物学报,2007,33(10)：1637-1643.
110. Jacobs DI, Vander HR, Verpoorte R. Proteomics in plant biotechnology andsecondary metabolism research[J]. Phytochemical Analysis,2000,11(5):277-287.
111. Gorg A, Obermaier C, Boguth G, et al. The current state of two-dimensional electrophoresis with immobilized pH gradients[J]. Electrophoresis,2000,21(6):1037-1053.
112. Kolli VSK, Orlando R. Complete sequence confirmation of large polypeptides by high energy collisional activation of multiply protonated ions[J]. J. Am. Soc. Mass Spectrom,1995,6(4): 234-241.
113. Preisig C, Byng G. Applications of mass spectrometry in screening for new biocatalysts[J]. J Mol Catal B Enzymatic,2001,11(4):733-741.
114. Fourgoux-Nicol A, Drouaud J, Haouazine N, et al. Isolation of rapeseed genes expressed early and specifically during development of the male gametophyte[J]. Plant Mol Biol,1999,40(5):857-872.
1. Anderson NL, Anderson NG. Proteome and proteomics:new technologies, new concepts, and new words[J]. Electrophoresis,1998,19(11):1853-1861.
2. Tyer M, Mann M. From genomics to proteomics[J]. Nature,2003,422(6928):193-197.
3. O'Farrell PH. High resolution two-dimensional electrophoresis of proteins[J]. The Journal of Biological Chemistry,1975,250(10):4007-4021.
4. Graves DA, Stewart J. Analysis of the protein constituency of developing cotton fibers[J]. J. exp. Bot, 1988,39(198):59-69.
5. Turley RB, Ferguson DL. Changes of ovule proteins during early fiber development in a normal and a fiberless line of cotton (Gossypium hirsutum L.) [J]. Journal of Plant Physiology,1996,149(6): 695-702.
6. Imin N, Kerim T, Weinman JJ, et al. Characterization of rice anther proteins expressed at the young microspore[J]. Proteomics,2001,1(9):1149-1161.
7. Wei W, Vignani R, Scali M, et al.A universal and rapid protocol for protein extraction from recalcitrant plant tissues for proteomic analysis[J]. Electrophoresis,2006,27(13):2782-2786.
8. Gorg A, Obermaier C, Boguth G,et al. Recent developments in two-dimensional electrophoresis with immobilized pH gradients:wide ph gradients up to pH 12, longer separation distances and simplified procedures[J]. Electrophoresis,1999,20(4-5):712-717.
9.刘康,胡凤萍,张天真.棉花胚珠与纤维蛋白质的两种提取方法比较研究[J].棉花学报,2005,17(6)：323-327.
10. Cremer F, Van de Walle C. Method for extraction of proteins from green plant tissues for two-dimensional polyacrylamide gel electrophoresis. Analytical Biochemistry,1985,147(1):22-26.
11. Nandakumar MP, Shen J, Raman B, et al. Solubilization of trichloroacetic acid (TCA) precipitated microbial proteins via NaOH for two-dimensional electrophoresis[J]. Journal of Proteome Research, 2003,2(1):89-93.
12. Gorg A, Obermaier C, Boguth G,et al. The current state of two-dimensional electrophoresis with immobilized pH gradients[J]. Electrophoresis,2000,21(6):1037-1053.
13.席景会,殷建文,岳琳,等.拟南芥全细胞蛋白质样品制备及其双向电泳条件的优化[J].吉林大学学报(理学版),2006,44(6)：1011-1014.
14.刘伟霞，潘映红.适用于小麦叶片蛋白质组分析的样品制备方法[J].中国农业科学,2007,40(10)：2169-2176.
1.史典义,赵晓菊,周正富,等.植物细胞核雄性不育的分子机制[J].植物生理学通讯,2007,43(3)：556-562.
2.朱四元,陈金湘.棉花雄性不育系的选育及不育机理的研究进展[J].江西棉花,2004,26(1)：11-15.
3. Tyer M, Mann M. From genomics to proteomics[J]. Nature,2003,422(6928):193-197.
4.文李,刘盖,张再君,等.红莲型水稻细胞质雄性不育花药蛋白质组学初步分析[J].遗传,2006,28
(3)：311-316.
5.曾维英,杨守萍,盖均镒,等.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[J].中国农业科学,2007,40(12)：2679-2687.
6. Wei W, Vignani R, Scali M, et al. A universal and rapid protocol for protein extraction from recalcitrant plant tissues for proteomic analysis[J]. Electrophoresis,2006,27(13):2782-2786.
7. Shan SW, Tang MK, Chow PH, et al. Induction of growth arrest and polycomb gene expression by reversine allows C2C12 cells to be reprogrammed to various differentiated cell types[J]. Proteomics, 2007,7(23):4303-4316.
8.豆丽萍,王庆亚,唐灿明.陆地棉双隐性核雄性不育系ms5ms6花药发育过程的研究[J].棉花学报,2009,21(4)：265-270.
9. Ach RA, Durfee T, Miller AB, et al. RRB1 and RRB2 encode maize retinoblastoma-related proteins that interact with a plant D-type cyclin and geminivirus replication protein[J]. Mol Cell Biol,1997, 17(9):5077-5086.
10. Grafi G, Burrnett RJ, Helentjaris T, et al. A maize cDNA encoding a member of the retinoblastoma protein family-involvement in endoreduplication[J]. Proc Natl Acad Sci USA,1996,93(17): 8962-8967.
11. Nakagami H, Sekine M, Murakami H,et al. Tobacco rentinoblastoma-related protein phosophorylated by a distinct cyclin-dependent kinase complex with Cdc2/cyclin D in vitro[J]. Plant J,1999,18:243-252.
12. Vandepoele K, Raes J, De Veylder L, et al. Genome-wide analysis of core cell cycle genes in Arabidopsis[J]. Plant Cell,2002,14(4):903-916.
13. Gutierrez C. The retinoblastoma pathway in plant cell cycle and development[J]. Curr Opin Plant Biol,1998,1(6):492-497.
14.刘祚昌,李继耕,罗会馨,等.二磷酸核酮糖羧化酶与细胞质雄性不育性的研究[J].遗传学报,1983,10(1)：36-42.
15.夏凯,肖翊华,刘文芳.湖北光敏感核不育水稻光敏感期叶片中ATP含量与RuBPcase活力 的分析[J].杂交水稻,1989,30(4)：41-42.
16.韩鹰,陈刚,王忠.Rubisco活化酶的研究进展[J].植物学通报,2000,17(4)：306-311.
17.王幼宁,刘孟雨,李霞.植物3-磷酸甘油醛脱氢酶的多维本质[J].西北植物学报,2005,25(3)：607-614.
18.夏其昌,曾嵘.蛋白质化学与蛋白质组学[M].北京：科学出版社,2004(第一版)：311-335.
19. Silverstone AL, Sun TP. Gibberellins and green revolution[J]. Trends in Plant Science,2000,5(1): 1-2.
20. Hedden P, Kamiya Y. Gibberellin biosynthesis:enzymes, genes and their regulation[J]. Annu Rev Plant Physiol Plant Mol Biol,1997,48:431-460.
21.王伟,朱平,程克棣.植物赤霉素生物合成和信号传导的分子生物学[J].植物学通报,2002,19(2)：137-149.
22.刘忠松.不育花药的生理生化研究进展与展望[J].植物生理学通讯,1987,(2)：16-21.
23. Bonghi C, Rascio N, Ramina A, et al. Cellulase and polygalacturonase involvement in the abscission of leaf and explants of peach[J]. Plant Mol.Biol,1992,20(5):839-848.
24. Meakin PJ, Roberts JA. Anatomical and biochemical changes associated with the induction of oilseed rape pod dehiscence by Dasineura brassicae[J].Ann.Bot,1991,67(3):193-197.
25. Pressey R. Polygalacturonase in tree pollens[J]. Phytochemistry,1991,30(6):1753-1755.
26. Sitrit Y, Downie B, Bennett AB, et al. A novel exo-polygalacturonase is ass ociated with radicle protrusion in tomato seeds[J]. Plant Physiol,1996, 111(Supple ment):161-163.
27. Lassner MW, Lardizabal K, Metz JG A jojoba beta-Ketoacyl-CoA synthase cDNA complements the canola fatty acid elongation mutation in transgenic plants[J]. Plant Cell,1996,8(2):281-292.
28. Millar AA, Kunst L. Very-long-chain fatty acid biosynthesis is controlled through the expression and specificity of the condensing enzyme[J]. Plant J,1997,12(1):121-131.
29. James DW, Lim E, Keller J, et al. Directed tagging of the Arabidopsis FATTY ACID ELONGATION1 (FAE1) gene with the maize transposon activator[J]. Plant Cell,1995,7(3): 309-319.
30. Rossak M, Smith M, Kunst L. Expression of the FAE1 gene and FAE1 promoter activity in developing seeds of Arabidopsis thaliana[J]. Plant Mol Biol,2001,46(6):717-725.
31. Kunst L, Taylor DC, Underhill EW. Fatty acid elongation in developing seed of Arabidopsis thaliana[J]. Plant Physiol Biochem,1992,30(4):425-434.
32. Todd J, Post-Beittenmiller D, Jaworski JG. KCS1 encodes a fatty acid elongase 3-ketoacyl-CoA synthase affecting wax biosynthesis in Arabidopsis thaliana[J]. Plant J,1999,17(2):119-130.
33. Clemens S, Kunst L. Patent cooperation treaty int. Patent Appl. WO 0107586.2001.
34. Millar AA, Clemens S, Zachgo S, et al. CUT1, an Arabidopsis gene required for cuticular wax biosynthesis and pollen fertility, encodes a very-long-chain fatty acid condensing enzyme[J]. Plant Cell,1999,11(5):825-838.
35. Fourmann M, Barret P, Renard M, et al. The two genes homologous to Arabidopsis FAE1 co-segregate with the two loci governing erucic acid content in Brassica napus[J]. Theor Appl Genet, 1998,96(6):852-858.
36. Moon H, Smith MA, Kunst L. A condensing enzyme from the seeds of Lesquerella fendleri that specifically elongates hydroxy fatty acids[J]. Plant Physiol,2001,127(4):1635-1643.
37. Kajikawa M, Yamaoka S, Yamato KT, et al. Functional analysis of a beta-ketoacyl-CoA synthase gene, MpFAE2, by gene silencing in the liverwort Marchantia polymorpha L[J]. Biosci Biotechnol Biochem,2003,67(3):605-612.
38. Trenkamp S, Martin W, Tietjen K. Specific and differential inhibition of very-long-chain fatty acid elongases from Arabidopsis thaliana by different herbicides[J]. Proc Natl Acad Sci USA,2004, 101(32):11903-11908.
39. Paul S, Gable K, Beaudoin F, et al. Members of the Arabidopsis FAE1-like 3-ketoacyl-coA synthase gene family substitute for the elop proteins of Saccharomyces cerevisiae[J]. J Biol Chem,2006, 281(14):9018-9029.
40. Yephremov A, Wisman E, Huijser P, et al. Characterization of the FIDDLEHEAD gene of Arabidopsis reveals a link between adhesion response and cell differentiation in the epidermis[J]. Plant Cell,1999,11(11):2187-2201.
41. Pruitt RE, Vielle-Calzada JP, Ploense SE, et al. FIDDLEHEAD, a gene required to suppress epidermal cell interactions in Arabidopsis, encodes a putative lipid biosynthetic enzyme[J]. Proc Natl Acad Sci USA,2000,97(3):1311-1316.
42. Costaglioli P, Joubes J, Garcia C, et al. Profiling candidate genes involved in wax biosynthesis in Arabidopsis thaliana by microarray analysis[J]. Biochim Biophys Acta,2005,1734(3):247-258.
43. Poovaiah BW, Xia M, Liu Z, et al. Developmental regulation of the gene for chimeric calium/calmodulin-dependentprotein kinase in anthers[J]. Planta,1999,209(2):161-171.
