鹅掌楸叶片发育相关蛋白的双向电泳分析
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摘要
本文对ISO-DALT进行了优化，建立了适合木本植物叶片蛋白质分析的双向电泳技术体系，实验结果如下：
(1)三氯乙酸俩酮制干粉提取法是鹅掌楸叶片蛋白样品处理的较为优化的方法。
(2)确立了一套较为优化的等电聚焦程序，在相对密闭的条件下，可以减少电泳系统对CO_2的吸收，同时采用最佳的等电聚焦时间，使阴极漂移的现象大大减弱。
(3)平衡的时间以25-30分钟为佳，测量pH梯度时标样法要优于浸泡法。
从本文的电泳图谱可以发现，ISO-DALT技术体系也能够得到比较稳定的电泳图谱，完全可以达到实验的要求。
利用优化的ISO-DALT技术，对鹅掌楸叶片生长发育过程进行了双向电泳分析，发现：
(1)鹅掌楸叶片蛋白质斑点的分子量范围是12.7kD-101.8kD，等电点范围为3.2-7.9，绝大部分斑点主要集中于分子量范围为13.5kD-82kD，等电点范围为3.8-7.0的区域内。大部分蛋白质在叶片不同发育阶段中都稳定表达，有一些蛋白质具有发育阶段表达特异性。
(2)蛋白斑点(56.5kD，pI3.6)和(28kD，pI3.3)在叶片迅速扩展阶段特异出现，这些蛋白质与此发育阶段密切相关。而蛋白斑点(39.9kD，pI5.0)、(39.9kD，pI5.12)、(38.5kD，pI5.0)和(38.5kD，pI5.12)从迅速扩展期到成熟期逐渐消失，且消失过程具有同步性。
(3)5月到6月的成熟期叶片蛋白质变化比较明显，蛋白斑点(53kD，pI5.95)的丰度明显下调，与黄化期蛋白质变化相比较后可以发现，斑点(18.6kD，pI4.73)、(20.1kD，pI5.90)、(13.7kD，pI3.80)、(13.7kD，pI4.09)和(13.7kD，pI4.37)的丰度上调与点(53kD，pI5.95)的丰度下调密切相关，这些蛋白在蛋白质周转中可能起着重要的作用。
(4)蛋白斑点(24.5 kD，pI6.30)为叶片黄化期特异斑点，与该发育阶段密切相关。
Some improvement adapting to the analysis of the proteins of woody plant in ISO-DALT was made and the results were as follows:
First, dry powder made by trichloroacetic acid and acetone was the better method in all of four disposal methods of leaf. Second, in this modified IEF protocol, a hermetic isoelectrofocusing system was set up to inhibit CO2 absorption and the cathode drift was also restrained. Furthermore, the right equilibrium time was 25 to 30 minutes and the method measuring pH grads by marker was better than that by marinating. In conclusion, all the patterns of ISO-DALT showed that the differentiation powder of the modified IEF protocol was improved greatly to meet the needs of ordinary biochemistry labs.
The modified method was applied to analyze the leaf proteins related to development of Liriodendron. It was found that abundant protein spots of leaf were visualized in 2-DE gel and most of them kept the same. Their isoelectric point were found to lie between 3.2 and 7.9,and most of them were in the range of 3.8-7.0.Their molecular weights were from 12.7 to 101.8 kD, and most of them were in the range of 13.5 ~ 82 kD. Most of protein spots expressed stably in the different development of leaf, but some of them were differential.
(56.5kD, pI3.6) and (28kD, pD.3) appearing at rapid enlarging stage of leaf had consanguineous relationship with development stage. (39.9kD, pI5.0) , (39.9kD, pI5.12) , (38.5kD, pI5.0) and ( 38.5kD, pI5.12) disappeared gradually from rapid enlarging stage to mature stage.
2-DE gel of mature leaf had evident variety from May to June, just as the variety of etiolation stage. The increase of richness of (18.6 kD, pI4.73 ), (20.1 kD, pI5.90 ), (13.7 kD, pD.80) , (13.7 kD, pI4.09) and (13.7 kD, pI4.37) which might have an important effect on proteins' turnover had close to the decline of the richness of (53 kD, pI5.95 ) .
The protein spot (24.5 kD, pI6.30) which had close relationship with etiolation stage should be the specific spot of etiolation stage of leaf.
(1)三氯乙酸俩酮制干粉提取法是鹅掌楸叶片蛋白样品处理的较为优化的方法。
(2)确立了一套较为优化的等电聚焦程序，在相对密闭的条件下，可以减少电泳系统对CO_2的吸收，同时采用最佳的等电聚焦时间，使阴极漂移的现象大大减弱。
(3)平衡的时间以25-30分钟为佳，测量pH梯度时标样法要优于浸泡法。
从本文的电泳图谱可以发现，ISO-DALT技术体系也能够得到比较稳定的电泳图谱，完全可以达到实验的要求。
利用优化的ISO-DALT技术，对鹅掌楸叶片生长发育过程进行了双向电泳分析，发现：
(1)鹅掌楸叶片蛋白质斑点的分子量范围是12.7kD-101.8kD，等电点范围为3.2-7.9，绝大部分斑点主要集中于分子量范围为13.5kD-82kD，等电点范围为3.8-7.0的区域内。大部分蛋白质在叶片不同发育阶段中都稳定表达，有一些蛋白质具有发育阶段表达特异性。
(2)蛋白斑点(56.5kD，pI3.6)和(28kD，pI3.3)在叶片迅速扩展阶段特异出现，这些蛋白质与此发育阶段密切相关。而蛋白斑点(39.9kD，pI5.0)、(39.9kD，pI5.12)、(38.5kD，pI5.0)和(38.5kD，pI5.12)从迅速扩展期到成熟期逐渐消失，且消失过程具有同步性。
(3)5月到6月的成熟期叶片蛋白质变化比较明显，蛋白斑点(53kD，pI5.95)的丰度明显下调，与黄化期蛋白质变化相比较后可以发现，斑点(18.6kD，pI4.73)、(20.1kD，pI5.90)、(13.7kD，pI3.80)、(13.7kD，pI4.09)和(13.7kD，pI4.37)的丰度上调与点(53kD，pI5.95)的丰度下调密切相关，这些蛋白在蛋白质周转中可能起着重要的作用。
(4)蛋白斑点(24.5 kD，pI6.30)为叶片黄化期特异斑点，与该发育阶段密切相关。
Some improvement adapting to the analysis of the proteins of woody plant in ISO-DALT was made and the results were as follows:
First, dry powder made by trichloroacetic acid and acetone was the better method in all of four disposal methods of leaf. Second, in this modified IEF protocol, a hermetic isoelectrofocusing system was set up to inhibit CO2 absorption and the cathode drift was also restrained. Furthermore, the right equilibrium time was 25 to 30 minutes and the method measuring pH grads by marker was better than that by marinating. In conclusion, all the patterns of ISO-DALT showed that the differentiation powder of the modified IEF protocol was improved greatly to meet the needs of ordinary biochemistry labs.
The modified method was applied to analyze the leaf proteins related to development of Liriodendron. It was found that abundant protein spots of leaf were visualized in 2-DE gel and most of them kept the same. Their isoelectric point were found to lie between 3.2 and 7.9,and most of them were in the range of 3.8-7.0.Their molecular weights were from 12.7 to 101.8 kD, and most of them were in the range of 13.5 ~ 82 kD. Most of protein spots expressed stably in the different development of leaf, but some of them were differential.
(56.5kD, pI3.6) and (28kD, pD.3) appearing at rapid enlarging stage of leaf had consanguineous relationship with development stage. (39.9kD, pI5.0) , (39.9kD, pI5.12) , (38.5kD, pI5.0) and ( 38.5kD, pI5.12) disappeared gradually from rapid enlarging stage to mature stage.
2-DE gel of mature leaf had evident variety from May to June, just as the variety of etiolation stage. The increase of richness of (18.6 kD, pI4.73 ), (20.1 kD, pI5.90 ), (13.7 kD, pD.80) , (13.7 kD, pI4.09) and (13.7 kD, pI4.37) which might have an important effect on proteins' turnover had close to the decline of the richness of (53 kD, pI5.95 ) .
The protein spot (24.5 kD, pI6.30) which had close relationship with etiolation stage should be the specific spot of etiolation stage of leaf.
引文
1 Abe,T.,Gusti,R.S.,Ono,M.and Sasahara,T.Variations in glutelin and high molecular weight endosperm proteins among subspecies of rice(Oryza sativa L.) detected by6 two-dimensional gel electrophoresis.Genes Genet.Syst. 1996,71:63～68
2 Agrawal. G. K.,R. Rakwal, M. Yonekura, A. Kubo & H. Saji. Proteome analysis of differentially displayed proteins as a tool for investigating ozone stress in rice(Oryza sativa L.) seedlings.Proteomics.2002,2:947～959
3 Antherton J G, Yen D M, Tucker G A. Timing of the protein changes during phase transition in Cineraria J. J Hort Sci Viol, 1998,73(1):35～38
4 Araki T, Komeda Y.Electrophoretic Analysis of Florally-Evoked Meristems of Pharbitis nil Choisy cv.Violet.Plant Cell Physiol, 1990, 31: 137
5 Bahrman,N.,Zivy, M.,Damerval,C.and Baradat,P.Organisation of the variability of abundant proteins in seven geographical origins of maritime pine (Pinus pinaster Ait). Theor.Appl.Genet. 1994,88:407～411
6 Bahrman,N.and Petit,R.Genetic polymorphisms in maritime pine (Pinus pinaster Ait) assessed by two-dimensional gel electrophoresis of needle,bud and pollen proteins.J.Mol.Evol. 1995,41:231～237.
7 Barreneche,T.,Bahrman,N.and Kremer, A.Two dimensional gel electroresis confirms the low level of genetic differentiation between Qrercus robur and Quercus petraea(Matt) Liebl.Forest Genetic. 1996,3: 89～92
8 Bestel-Corre,G.,E.Dumas-Gaudot,V.Poinsot,M.Kieu,J.F.Dierick,T.D.J.van Remacle,V.Gianinazzi-Pearson & S.Gianinazzi.Proteome analysis and identification of symbiosis-related proteins from Medicago truncatula Gaertn.by two-dimensional electrophoresis and mass spectrometry.Electrophoresis.2002,3:89～92
9 Burstin,J.,de Vienne,D.,Dubreuil,P.and Damerval,C.Molecular markers and protein quantities as genetic descriptors in maize.I.Genetic diversity among 21 inbred lines. Theor.Appl.Genet.1994,89:943～950
10 Chlan CA,Dure L.Plant seed embryogensis as a tool for molecular biology.Mol.Cell Biochem, 1983, 55:5
11 David J.L.,Zivy M.,Cardin M.L.and Brabant,P.Protein evolution in dynamically managed populations of wheat:adaptative responses to macro-environmental conditions. Theor. Appl. Genet., 1997,95:932～941
12 de Castro LAB et al.Spitial and Temporal Gene Rxpression Patterns Occur during Corm Development.The Plant Cell, 1992,4:1549
13 De Venne,D., Burstin,J.,Gerber, S.,Leonardi,A.,Le Guilloux,M., Murigeneux, A., Beckert, M., Bahrman,N.,Damerval,C.and Zivy, M.Two-dimensional electrophoresis of proteins as a source of monogenic and codominant markers for population genetics and mapping
the expressed genome.Heredity 1996,76:166～177
14 Dure L et al. Developmental Biochemistry of cottonseed Embryogensis and Germination: Changing Messenger Ribonucleic Acid Populations As shown by in vitro and in vitro Protein Sythesis. Biochem,1981, 20:4162
15 Geltz N R, Russell S D. Two-dimensional electrophoretic studies of the proteins and polypeptides in mature pollen grain and the male germ unit of plumage zeylanica.Plant Physiol, 1988,88:764～769
16 Gorg,A.,Postel,W.,Baumer, M.and Weiss,W.Two-dimensional polyacrylamide gel electrophoresis,with immobilibed pH gradients in the first dimension,of barley seed proteins:descrimination of cultivars with different malting grades. Electrophoresis. 1992,13:192～203
17 Gorg,A.,Postel,W.,Domscheit,A.and Gunther, S. Two-dimensional electro-phoresis with immobilized pH gradients of leaf proteins from barely (Hordeum vulgare): method,reproducibility and genetic aspects. Electrophoresis. 1988,9:681～692
18 Granier, F. Extraction of plant proteins for two-dimensional electrophoresis. Electrophoresis, 1988, 9:712～718
19 Halloway P, Arundel P.High-resolution two-dimensional electrophoresis of plant proteins. Anal Biochem, 1988, 172:33～36
20 Humphery-Smith,I.,Cordwell,S.J. and Blackstock,W.P. Proteome research: complementarity and limitations with respect to the RNA and RNA worlds. Electrophoresis, 1997,18:1217～1242
21 Klose J, Kobalz U.New approaches to the evaluation of abnormal embryonic development.Electrophoresis,1995,16:1034～1059
22 Kvistine Vander Mijnsbrugge,Hugo Meyermans,Marc Van Montagu,Guy Bauw,Wout Boerjan.Wood formation in poplar: identification, characterization, and seasonal variation of xylem proteins.Planta,2000,210:589～598
23 Leonardi,A.,Damerval,D and de Vienne,D.Organ-specific variability and inheritance of maize proteins revealed by two-dimensional electrophoresis. Genet. Res. Camb. 1988, 52:97～103
24 LI Zhou-Qi,WANG Zhang-Rong.Relationship Between Heterosis and Endogenous Plant Hormones in Liriondendron[J]. Acta Botanica Sinica,2002,44(6):698～701
25 O'Farrell. High resolution two-dimensional electrophoresis of proteins. The Journal of Biological Chemistry, 1975,250(10):4007～4021
26 Picard, P., Bourgoin-Greneche, M. and Zivy, M. Potential of two-dimensional electrophoresis in routine identification of closely related durum wheat lines.Electrophoresis 1997,18:174～181
27 Posch,A.,van den Berg,B.M.,Duranton,C.and Gorg,A.Polymorphism of pepper (Capsicum annuum L.) seed proteins studied by 2-D electrophoresis with immobilized
pH gradients:methodical and genetic aspects. Electrophoresis. 1994,15:297～304
28 Posch,A.,van den Berg,B.M.,Postel,W.and Gorg,A.Gentic variability of pepper (Capsicum annuum L.) seed proteins studied by 2-D electrophoresis with immobilized pH gradients.Electrophoresis.1992,13:774～777
29 Ramagopal,S.K.Protein polymorphism in sugarcane revealed by two-dimensional gel analysis. Theor.Appl.Genet. 1990,79:297～304
30 Ramani.S. & S.K.Apte.Transient expression of multiple genes in salinity-stressed young seedling of rice(Oryza sativa L.cv.Bura Rata).Biochemical and Bilphysical Research Communications, 1997,13:663～667
31 Salekdeh,G.H.,J.Siopongco,L.J.Wade,B.Ghareyazie & J.Bennett.Proteomic analysis of rice leaves during drought stress and recovery.Proteomics.2002,2:1131～1145
32 Saruyama,H.and Shinbashi,N.Identification of specific proteins from seed embryos by two-dimensional gel electrophoresis for the discrimination between indica and japonica rice.Theor.Appl. Genet. 1992,84:947～951
33 Shigetoshi Suzuki,Hitoshi Nakamoto,Maurice S.B.Ku,and Gerald E.Edwards. Influence of leaf age on Photosynthesis,Enzyme Activity, and Metabolite levels in Wheat.Plant Physiol, 1987,84:1244～1248
34 Snowball A M, Zeman A M, Tchan Y T, et al. Phase change in cirus: immunologically detectable differences between juvenile and mature plants J. Austr Plant physiol, 1991,18:385～396
35 Stanley Fields.Proteomics in genomeland,Science, 2001,291:1221
36 Tsugita,A.,T.Kawakami,Y.Uchiyama,M.Kamo,N.Miyatake & Y.Nozu.Separation and characterization of rice proteins.Electrophoresis, 1994,15:708～720
37 Wang C S, Walling L L, Eckard K J. Pattern accumulation in developing anthers of lillium longiflorum correlate weth histological events.Amer J Bot 1992,79:118～127
38 Wilkins,M.R.,Williams,K.L.,Appel,R.D.,and Hochstrasser, D.F.Proteome Research: New Frontiers in Functional Genomics. Spinger, Berlin, 1997,p.243
39 Wilson Abbott.And now for the proteome,Nature, 2001,409:747
40 Woolhouse H W, In:The Molecular Biology of Plant Development.Eds: Smith H, Grierson D, Blackwell Scientific Publications, Oxford, London1982:256～281
41 Wu F S,Wang M Y.Extraction of proteins for sodium dodecyl sulfate-polyacrylamide gel electrophoresis from protease-rich plant tissues.Anal Biochem, 1984, 139:704～712
42 Zivy, M.,Thiellement,H.,de Vienne,D.and Hofmann,J.-p.Study on nuclear and cytoplasmic genome expression in wheat by two-dimensional gel electrophoresis. Ⅰ.First results on 18 alloplasmic lines. Theor.Appl. Genet. 1983,66:1～7
43 Zivy, M.,Thiellement,H.,de Vienne,D.and Hofmann,J.-p.Study on nuclear and cytoplasmic genome expression in wheat by two-dimensional gel electrophoresis. Ⅱ.Genetic differences between two lines and two groups of cytoplasms at five
developmental stages or organs. Theor.Appl.Genet. 1984,68:335～345
44 陈军营，刘万代，邰国琴，杜申焕．植物特异蛋白的研究进展[J]．河南农业大学学报，1999，33(1)：5～10
45 陈荣智，翁清妹，黄臻，祝莉莉，何光存．水稻对褐飞虱抗性相关蛋白的双向电泳分析[J]．植物学报，2002，44(4)：427～432
46 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
47 陈伟，吕柳新，黄春梅，周洁，梁文裕．‘乌叶’荔枝胚胎发育过程特异蛋白的变化[J]．园艺学报，2001，28(6)：504～508
48 董长江，郑常文，刘丙辰等．诸葛菜精细胞特异蛋白多肽的研究[J]．植物学报，1997，39(8)：712～716
49 樊汝汶，尹增芳，尤录祥．中国鹅掌楸花芽分化的细胞形态学观察[J]．南京林业大学学报，1990，14(2)：26～32．
50 樊汝汶，尹增芳．中国鹅掌楸花粉母细胞减数分裂的超微结构观察[J]．南京林业大学学报，1992，16(2)：31～36．
51 樊汝汶，方炎明，黄金生．鹅掌楸属植物引导组织和花粉管生长[J]．西北植物学报，1995，15(3)：219～224．
52 樊汝汶，叶建国，尹增芳．鹅掌楸种子和胚胎发育的研究[J]．植物学报，1992，34(6)：437～442．
53 范国强，蒋建平．泡桐叶片蛋白质提取方法的研究[J]．植物学通报，1997，14(3)：61～64
54 范国强，李有，郑建伟，翟晓巧．泡桐丛枝病发生相关蛋白质的电泳分析[J]．林业科学，2003，39(2)：119～122
55 方炎明．中国鹅掌楸的地理分布与空间格局[J]．南京林业大学学报，1994，18(2)：13～18．
56 谷瑞升，蒋湘宁，裴东．胡杨愈伤组织继代增殖和器官发生中蛋白分子标记的研究[J]．林业科学，2002，38(5)：1～6
57 谷瑞升，刘群录，陈雪梅，蒋湘宁．一种省时高效的木本植物蛋白双向电泳分析方法．北京林业大学学报[J]，1999，21(5)：7～10
58 哈密斯 B D，利克五德 D．蛋白质凝胶电泳实践[M]．北京，科学出版社，1986
59 郭尧君．蛋白质电泳实验技术[M]．北京．科学出版社，1999．142
60 郝日明，善安，汤诗杰等鹅掌楸在中国的自然分布及其特点[J]．植物资源与环境，1995，4(1)：1～6．
61 何瑞锋．水稻“斑马叶”突变性状的初步研究．武汉大学，硕士学位论文，2000
62 贺福初．蛋白质组(proteome)研究——后基因组时代的生力军[J]．科学通报，1999，44(2)：113～123
63 贺力强．双向电泳及其在水稻(Oryza sativa L)细胞质雄性不育相关蛋白质研究中的应用．湖南农业大学，硕士学位论文，2001
64 贺善安，郝日明，汤诗杰．鹅掌楸致濒的生态因素研究[J]．植物资源与环境，1996，5(1)：1～8
65 胡旺顺．IEF／SDS-PAGE技术及其在光温敏雄性不育水稻育性相关蛋白分析中的应用研究．湖南农业大学，博士学位论文，2001
66 黄海，植物叶发育调控机理研究的进展[J]．植物学通报，2003，20(4)：416～422
67 黄敏仁，陈道明．杂种马褂木的同工酶分析[J]．南京林产工业学院学报，1979，(1，2)：156～158．
68 黄双全，郭友好，陈家宽等．用RAPD方法初探鹅掌楸的花粉流[J]．科学通报，1998，43(14)：1517～1519．
69 黄双全，郭友好，吴艳等．鹅掌楸的花部数量变异与结实率[J]．植物学报，1998，40(1)：22～27．
70 李周岐，王章荣．RAPD标记在鹅掌楸属中的遗传研究[J]．林业科学，2002，38(1)：150～153
71 林金科，郑金贵，袁明，张学琴，王凤．茶树蛋白质提取及双向电泳的改良方法[J]．茶叶科学，2003，23(1)：16～20
72 刘洪谔，沈湘林，曾玉亮．中国鹅掌楸、美国鹅掌楸及其杂种在形态和生长上的遗传变异[J]．浙江林业科技，1991，11(5)：18～22．
73 刘西俊，周丕振，王淑燕等．鹅掌楸生理特性及适应性研究[J]．西北植物学报，1989，9(3)：183～190．
74 南京林产工业学院林学系育种组．亚美杂种马褂木的育成[J]．林业科技通讯，1973，(12)：10～11．
75 钱小红等．蛋白质组学：从序列到功能[M]．科学出版社，2002
76 饶桂荣，杨继华，薛妙男．沙田柚各器官特异蛋白质的研究[J]．广西师范大学学报(自然科学版)，2000, 18(2)：78～81
77 孙崇荣，李育庆，汪景长等．水稻种子蛋白质组分的双向电泳分析[J]．作物学报．1987，13(91)：63～67
78 谭克辉．春化过程中特异蛋白质的合成[J]．植物学集刊，1992，6：13～15
79 汤健，朱广廉，曹宗巽．兰州百合精细胞特异蛋白的研究[J]．植物学报，1997，39(6)：511～516
80 万晶宏，贺福初．蛋白质组技术的研究进展[J]．科学通报，1999，44(9)：904～912
81 王锡锋，周广和．大麦黄矮病毒介体麦二叉蚜和麦长管蚜体内传毒相关蛋白的确定[J]．科学通报，2003，48(15)：1671～1675
82 王章荣．中国马褂木遗传资源的保护与杂交育种前景[J]．林业科技通讯，1997，(9)：8～10．
83 王振英，彭永康，陈宏．用双向电泳分析百合减数第一分裂周期蛋白质的变化[J]，2001，21(4)：596～599
84 王志珍，邹承鲁．后基因组——蛋白质组研究[J]．生物化学和生物物理学报，1998，30(6)：533～539
85 韦仲新，吴征益．鹅掌楸属花粉的超微结构研究及系统学意义[J]．云南植物研究，1993，15(2)：163～166．
86 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
87 杨继华，颜承，薛妙男，秦新民沙田柚花粉蛋白质双向电泳分析[J]．广西师范大学学报(自然科学版)[J]，2002，20(2)：73～77
88 叶金山，季孔庶，王章荣．杂种马褂木无性系插条生根能力的遗传变异[J]．南京林业大学学报，1998a，22(2)：71～74．
89 叶金山，王章荣．杂种马褂木杂种优势的遗传分析[J]．林业科学，2002，38(4)：67～71
90 叶金山．鹅掌楸杂种优势的生理遗传基础．南京林业大学博士学位论文，1998b．
91 尹增芳，樊汝汶．鹅掌楸花粉败育过程的超微结构观察[J]．植物资源与环境，1997，6(2)：1～7．
92 尹增芳，樊汝汶．中国鹅掌楸小孢子发生的细胞化学研究[J]．植物学通报，1998，15(3)：34～37
93 尹增芳，樊汝汶．中国鹅掌楸雄配子体发育的超微结构研究[J]．植物资源与环境，1994，3(1)：1～8．
94 张往祥，李群，曹福亮．杂交马褂木叶片发育过程中资源利用效率的变化格局[J]．植物资源与环境学报．2002，11(4)：9～14
95 张晓平，方炎明．杂种鹅掌楸不同季节扦插特征比较[J]．浙江林学院学报，2003，20(3)：249～253
96 张晓平，方炎明．杂种鹅掌楸插穗不定根发生与发育的解剖学观察[J]．植物资源与环境学报，2003，12(1)：10-15
97 周涵韬，林鹏．盐胁迫下红树植物蛋白质的比较分析[J]．海洋科学，2002，26(4)：5～7
98 周坚，樊汝汶．鹅掌楸属两种植物花粉品质和花粉管生长的研究[J]．林业科学，1994，30(5)：405～411．
99 周坚，樊汝汶．中国鹅掌楸雄性生殖单位的二维结构[J]．南京林业大学学报，1995，19(1)：15～20．
100 周坚，樊汝汶．中国马褂木精细胞分离技术[J]．南京林业大学学报，1999a，23(3)：21～24
101 周坚，樊汝汶．中国马褂木生活精细胞的分离与观察[J]．南京林业大学学报，1999b，23(1)：15～18
102 朱虹．T型小麦细胞质雄性不育和可育株总水溶性差异蛋白质研究[J]．东北农业大学，博士学位论文，2003
103 朱晓琴，贺善安，姚青菊等．鹅掌楸居群遗传结构及其保护对策[J]．植物资源与环境，1997，6(4)：7～14．
104 朱晓琴，马建霞，姚青菊等．鹅掌楸(Liriodendron chinese)遗传多样性的等位酶论证[J]．植物资源与环境，1995，4(3)：9～14．
105 朱友林，王建，余潮．植物蛋白质双向电泳及其考马斯亮兰-银染复合染色法[J]．南昌大学学报，1999，23(2)：101～105
2 Agrawal. G. K.,R. Rakwal, M. Yonekura, A. Kubo & H. Saji. Proteome analysis of differentially displayed proteins as a tool for investigating ozone stress in rice(Oryza sativa L.) seedlings.Proteomics.2002,2:947～959
3 Antherton J G, Yen D M, Tucker G A. Timing of the protein changes during phase transition in Cineraria J. J Hort Sci Viol, 1998,73(1):35～38
4 Araki T, Komeda Y.Electrophoretic Analysis of Florally-Evoked Meristems of Pharbitis nil Choisy cv.Violet.Plant Cell Physiol, 1990, 31: 137
5 Bahrman,N.,Zivy, M.,Damerval,C.and Baradat,P.Organisation of the variability of abundant proteins in seven geographical origins of maritime pine (Pinus pinaster Ait). Theor.Appl.Genet. 1994,88:407～411
6 Bahrman,N.and Petit,R.Genetic polymorphisms in maritime pine (Pinus pinaster Ait) assessed by two-dimensional gel electrophoresis of needle,bud and pollen proteins.J.Mol.Evol. 1995,41:231～237.
7 Barreneche,T.,Bahrman,N.and Kremer, A.Two dimensional gel electroresis confirms the low level of genetic differentiation between Qrercus robur and Quercus petraea(Matt) Liebl.Forest Genetic. 1996,3: 89～92
8 Bestel-Corre,G.,E.Dumas-Gaudot,V.Poinsot,M.Kieu,J.F.Dierick,T.D.J.van Remacle,V.Gianinazzi-Pearson & S.Gianinazzi.Proteome analysis and identification of symbiosis-related proteins from Medicago truncatula Gaertn.by two-dimensional electrophoresis and mass spectrometry.Electrophoresis.2002,3:89～92
9 Burstin,J.,de Vienne,D.,Dubreuil,P.and Damerval,C.Molecular markers and protein quantities as genetic descriptors in maize.I.Genetic diversity among 21 inbred lines. Theor.Appl.Genet.1994,89:943～950
10 Chlan CA,Dure L.Plant seed embryogensis as a tool for molecular biology.Mol.Cell Biochem, 1983, 55:5
11 David J.L.,Zivy M.,Cardin M.L.and Brabant,P.Protein evolution in dynamically managed populations of wheat:adaptative responses to macro-environmental conditions. Theor. Appl. Genet., 1997,95:932～941
12 de Castro LAB et al.Spitial and Temporal Gene Rxpression Patterns Occur during Corm Development.The Plant Cell, 1992,4:1549
13 De Venne,D., Burstin,J.,Gerber, S.,Leonardi,A.,Le Guilloux,M., Murigeneux, A., Beckert, M., Bahrman,N.,Damerval,C.and Zivy, M.Two-dimensional electrophoresis of proteins as a source of monogenic and codominant markers for population genetics and mapping
the expressed genome.Heredity 1996,76:166～177
14 Dure L et al. Developmental Biochemistry of cottonseed Embryogensis and Germination: Changing Messenger Ribonucleic Acid Populations As shown by in vitro and in vitro Protein Sythesis. Biochem,1981, 20:4162
15 Geltz N R, Russell S D. Two-dimensional electrophoretic studies of the proteins and polypeptides in mature pollen grain and the male germ unit of plumage zeylanica.Plant Physiol, 1988,88:764～769
16 Gorg,A.,Postel,W.,Baumer, M.and Weiss,W.Two-dimensional polyacrylamide gel electrophoresis,with immobilibed pH gradients in the first dimension,of barley seed proteins:descrimination of cultivars with different malting grades. Electrophoresis. 1992,13:192～203
17 Gorg,A.,Postel,W.,Domscheit,A.and Gunther, S. Two-dimensional electro-phoresis with immobilized pH gradients of leaf proteins from barely (Hordeum vulgare): method,reproducibility and genetic aspects. Electrophoresis. 1988,9:681～692
18 Granier, F. Extraction of plant proteins for two-dimensional electrophoresis. Electrophoresis, 1988, 9:712～718
19 Halloway P, Arundel P.High-resolution two-dimensional electrophoresis of plant proteins. Anal Biochem, 1988, 172:33～36
20 Humphery-Smith,I.,Cordwell,S.J. and Blackstock,W.P. Proteome research: complementarity and limitations with respect to the RNA and RNA worlds. Electrophoresis, 1997,18:1217～1242
21 Klose J, Kobalz U.New approaches to the evaluation of abnormal embryonic development.Electrophoresis,1995,16:1034～1059
22 Kvistine Vander Mijnsbrugge,Hugo Meyermans,Marc Van Montagu,Guy Bauw,Wout Boerjan.Wood formation in poplar: identification, characterization, and seasonal variation of xylem proteins.Planta,2000,210:589～598
23 Leonardi,A.,Damerval,D and de Vienne,D.Organ-specific variability and inheritance of maize proteins revealed by two-dimensional electrophoresis. Genet. Res. Camb. 1988, 52:97～103
24 LI Zhou-Qi,WANG Zhang-Rong.Relationship Between Heterosis and Endogenous Plant Hormones in Liriondendron[J]. Acta Botanica Sinica,2002,44(6):698～701
25 O'Farrell. High resolution two-dimensional electrophoresis of proteins. The Journal of Biological Chemistry, 1975,250(10):4007～4021
26 Picard, P., Bourgoin-Greneche, M. and Zivy, M. Potential of two-dimensional electrophoresis in routine identification of closely related durum wheat lines.Electrophoresis 1997,18:174～181
27 Posch,A.,van den Berg,B.M.,Duranton,C.and Gorg,A.Polymorphism of pepper (Capsicum annuum L.) seed proteins studied by 2-D electrophoresis with immobilized
pH gradients:methodical and genetic aspects. Electrophoresis. 1994,15:297～304
28 Posch,A.,van den Berg,B.M.,Postel,W.and Gorg,A.Gentic variability of pepper (Capsicum annuum L.) seed proteins studied by 2-D electrophoresis with immobilized pH gradients.Electrophoresis.1992,13:774～777
29 Ramagopal,S.K.Protein polymorphism in sugarcane revealed by two-dimensional gel analysis. Theor.Appl.Genet. 1990,79:297～304
30 Ramani.S. & S.K.Apte.Transient expression of multiple genes in salinity-stressed young seedling of rice(Oryza sativa L.cv.Bura Rata).Biochemical and Bilphysical Research Communications, 1997,13:663～667
31 Salekdeh,G.H.,J.Siopongco,L.J.Wade,B.Ghareyazie & J.Bennett.Proteomic analysis of rice leaves during drought stress and recovery.Proteomics.2002,2:1131～1145
32 Saruyama,H.and Shinbashi,N.Identification of specific proteins from seed embryos by two-dimensional gel electrophoresis for the discrimination between indica and japonica rice.Theor.Appl. Genet. 1992,84:947～951
33 Shigetoshi Suzuki,Hitoshi Nakamoto,Maurice S.B.Ku,and Gerald E.Edwards. Influence of leaf age on Photosynthesis,Enzyme Activity, and Metabolite levels in Wheat.Plant Physiol, 1987,84:1244～1248
34 Snowball A M, Zeman A M, Tchan Y T, et al. Phase change in cirus: immunologically detectable differences between juvenile and mature plants J. Austr Plant physiol, 1991,18:385～396
35 Stanley Fields.Proteomics in genomeland,Science, 2001,291:1221
36 Tsugita,A.,T.Kawakami,Y.Uchiyama,M.Kamo,N.Miyatake & Y.Nozu.Separation and characterization of rice proteins.Electrophoresis, 1994,15:708～720
37 Wang C S, Walling L L, Eckard K J. Pattern accumulation in developing anthers of lillium longiflorum correlate weth histological events.Amer J Bot 1992,79:118～127
38 Wilkins,M.R.,Williams,K.L.,Appel,R.D.,and Hochstrasser, D.F.Proteome Research: New Frontiers in Functional Genomics. Spinger, Berlin, 1997,p.243
39 Wilson Abbott.And now for the proteome,Nature, 2001,409:747
40 Woolhouse H W, In:The Molecular Biology of Plant Development.Eds: Smith H, Grierson D, Blackwell Scientific Publications, Oxford, London1982:256～281
41 Wu F S,Wang M Y.Extraction of proteins for sodium dodecyl sulfate-polyacrylamide gel electrophoresis from protease-rich plant tissues.Anal Biochem, 1984, 139:704～712
42 Zivy, M.,Thiellement,H.,de Vienne,D.and Hofmann,J.-p.Study on nuclear and cytoplasmic genome expression in wheat by two-dimensional gel electrophoresis. Ⅰ.First results on 18 alloplasmic lines. Theor.Appl. Genet. 1983,66:1～7
43 Zivy, M.,Thiellement,H.,de Vienne,D.and Hofmann,J.-p.Study on nuclear and cytoplasmic genome expression in wheat by two-dimensional gel electrophoresis. Ⅱ.Genetic differences between two lines and two groups of cytoplasms at five
developmental stages or organs. Theor.Appl.Genet. 1984,68:335～345
44 陈军营，刘万代，邰国琴，杜申焕．植物特异蛋白的研究进展[J]．河南农业大学学报，1999，33(1)：5～10
45 陈荣智，翁清妹，黄臻，祝莉莉，何光存．水稻对褐飞虱抗性相关蛋白的双向电泳分析[J]．植物学报，2002，44(4)：427～432
46 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
47 陈伟，吕柳新，黄春梅，周洁，梁文裕．‘乌叶’荔枝胚胎发育过程特异蛋白的变化[J]．园艺学报，2001，28(6)：504～508
48 董长江，郑常文，刘丙辰等．诸葛菜精细胞特异蛋白多肽的研究[J]．植物学报，1997，39(8)：712～716
49 樊汝汶，尹增芳，尤录祥．中国鹅掌楸花芽分化的细胞形态学观察[J]．南京林业大学学报，1990，14(2)：26～32．
50 樊汝汶，尹增芳．中国鹅掌楸花粉母细胞减数分裂的超微结构观察[J]．南京林业大学学报，1992，16(2)：31～36．
51 樊汝汶，方炎明，黄金生．鹅掌楸属植物引导组织和花粉管生长[J]．西北植物学报，1995，15(3)：219～224．
52 樊汝汶，叶建国，尹增芳．鹅掌楸种子和胚胎发育的研究[J]．植物学报，1992，34(6)：437～442．
53 范国强，蒋建平．泡桐叶片蛋白质提取方法的研究[J]．植物学通报，1997，14(3)：61～64
54 范国强，李有，郑建伟，翟晓巧．泡桐丛枝病发生相关蛋白质的电泳分析[J]．林业科学，2003，39(2)：119～122
55 方炎明．中国鹅掌楸的地理分布与空间格局[J]．南京林业大学学报，1994，18(2)：13～18．
56 谷瑞升，蒋湘宁，裴东．胡杨愈伤组织继代增殖和器官发生中蛋白分子标记的研究[J]．林业科学，2002，38(5)：1～6
57 谷瑞升，刘群录，陈雪梅，蒋湘宁．一种省时高效的木本植物蛋白双向电泳分析方法．北京林业大学学报[J]，1999，21(5)：7～10
58 哈密斯 B D，利克五德 D．蛋白质凝胶电泳实践[M]．北京，科学出版社，1986
59 郭尧君．蛋白质电泳实验技术[M]．北京．科学出版社，1999．142
60 郝日明，善安，汤诗杰等鹅掌楸在中国的自然分布及其特点[J]．植物资源与环境，1995，4(1)：1～6．
61 何瑞锋．水稻“斑马叶”突变性状的初步研究．武汉大学，硕士学位论文，2000
62 贺福初．蛋白质组(proteome)研究——后基因组时代的生力军[J]．科学通报，1999，44(2)：113～123
63 贺力强．双向电泳及其在水稻(Oryza sativa L)细胞质雄性不育相关蛋白质研究中的应用．湖南农业大学，硕士学位论文，2001
64 贺善安，郝日明，汤诗杰．鹅掌楸致濒的生态因素研究[J]．植物资源与环境，1996，5(1)：1～8
65 胡旺顺．IEF／SDS-PAGE技术及其在光温敏雄性不育水稻育性相关蛋白分析中的应用研究．湖南农业大学，博士学位论文，2001
66 黄海，植物叶发育调控机理研究的进展[J]．植物学通报，2003，20(4)：416～422
67 黄敏仁，陈道明．杂种马褂木的同工酶分析[J]．南京林产工业学院学报，1979，(1，2)：156～158．
68 黄双全，郭友好，陈家宽等．用RAPD方法初探鹅掌楸的花粉流[J]．科学通报，1998，43(14)：1517～1519．
69 黄双全，郭友好，吴艳等．鹅掌楸的花部数量变异与结实率[J]．植物学报，1998，40(1)：22～27．
70 李周岐，王章荣．RAPD标记在鹅掌楸属中的遗传研究[J]．林业科学，2002，38(1)：150～153
71 林金科，郑金贵，袁明，张学琴，王凤．茶树蛋白质提取及双向电泳的改良方法[J]．茶叶科学，2003，23(1)：16～20
72 刘洪谔，沈湘林，曾玉亮．中国鹅掌楸、美国鹅掌楸及其杂种在形态和生长上的遗传变异[J]．浙江林业科技，1991，11(5)：18～22．
73 刘西俊，周丕振，王淑燕等．鹅掌楸生理特性及适应性研究[J]．西北植物学报，1989，9(3)：183～190．
74 南京林产工业学院林学系育种组．亚美杂种马褂木的育成[J]．林业科技通讯，1973，(12)：10～11．
75 钱小红等．蛋白质组学：从序列到功能[M]．科学出版社，2002
76 饶桂荣，杨继华，薛妙男．沙田柚各器官特异蛋白质的研究[J]．广西师范大学学报(自然科学版)，2000, 18(2)：78～81
77 孙崇荣，李育庆，汪景长等．水稻种子蛋白质组分的双向电泳分析[J]．作物学报．1987，13(91)：63～67
78 谭克辉．春化过程中特异蛋白质的合成[J]．植物学集刊，1992，6：13～15
79 汤健，朱广廉，曹宗巽．兰州百合精细胞特异蛋白的研究[J]．植物学报，1997，39(6)：511～516
80 万晶宏，贺福初．蛋白质组技术的研究进展[J]．科学通报，1999，44(9)：904～912
81 王锡锋，周广和．大麦黄矮病毒介体麦二叉蚜和麦长管蚜体内传毒相关蛋白的确定[J]．科学通报，2003，48(15)：1671～1675
82 王章荣．中国马褂木遗传资源的保护与杂交育种前景[J]．林业科技通讯，1997，(9)：8～10．
83 王振英，彭永康，陈宏．用双向电泳分析百合减数第一分裂周期蛋白质的变化[J]，2001，21(4)：596～599
84 王志珍，邹承鲁．后基因组——蛋白质组研究[J]．生物化学和生物物理学报，1998，30(6)：533～539
85 韦仲新，吴征益．鹅掌楸属花粉的超微结构研究及系统学意义[J]．云南植物研究，1993，15(2)：163～166．
86 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
87 杨继华，颜承，薛妙男，秦新民沙田柚花粉蛋白质双向电泳分析[J]．广西师范大学学报(自然科学版)[J]，2002，20(2)：73～77
88 叶金山，季孔庶，王章荣．杂种马褂木无性系插条生根能力的遗传变异[J]．南京林业大学学报，1998a，22(2)：71～74．
89 叶金山，王章荣．杂种马褂木杂种优势的遗传分析[J]．林业科学，2002，38(4)：67～71
90 叶金山．鹅掌楸杂种优势的生理遗传基础．南京林业大学博士学位论文，1998b．
91 尹增芳，樊汝汶．鹅掌楸花粉败育过程的超微结构观察[J]．植物资源与环境，1997，6(2)：1～7．
92 尹增芳，樊汝汶．中国鹅掌楸小孢子发生的细胞化学研究[J]．植物学通报，1998，15(3)：34～37
93 尹增芳，樊汝汶．中国鹅掌楸雄配子体发育的超微结构研究[J]．植物资源与环境，1994，3(1)：1～8．
94 张往祥，李群，曹福亮．杂交马褂木叶片发育过程中资源利用效率的变化格局[J]．植物资源与环境学报．2002，11(4)：9～14
95 张晓平，方炎明．杂种鹅掌楸不同季节扦插特征比较[J]．浙江林学院学报，2003，20(3)：249～253
96 张晓平，方炎明．杂种鹅掌楸插穗不定根发生与发育的解剖学观察[J]．植物资源与环境学报，2003，12(1)：10-15
97 周涵韬，林鹏．盐胁迫下红树植物蛋白质的比较分析[J]．海洋科学，2002，26(4)：5～7
98 周坚，樊汝汶．鹅掌楸属两种植物花粉品质和花粉管生长的研究[J]．林业科学，1994，30(5)：405～411．
99 周坚，樊汝汶．中国鹅掌楸雄性生殖单位的二维结构[J]．南京林业大学学报，1995，19(1)：15～20．
100 周坚，樊汝汶．中国马褂木精细胞分离技术[J]．南京林业大学学报，1999a，23(3)：21～24
101 周坚，樊汝汶．中国马褂木生活精细胞的分离与观察[J]．南京林业大学学报，1999b，23(1)：15～18
102 朱虹．T型小麦细胞质雄性不育和可育株总水溶性差异蛋白质研究[J]．东北农业大学，博士学位论文，2003
103 朱晓琴，贺善安，姚青菊等．鹅掌楸居群遗传结构及其保护对策[J]．植物资源与环境，1997，6(4)：7～14．
104 朱晓琴，马建霞，姚青菊等．鹅掌楸(Liriodendron chinese)遗传多样性的等位酶论证[J]．植物资源与环境，1995，4(3)：9～14．
105 朱友林，王建，余潮．植物蛋白质双向电泳及其考马斯亮兰-银染复合染色法[J]．南昌大学学报，1999，23(2)：101～105