Two-dimensional Electrophoresis System for Cucumber Female Flower ’s Corolla

doi:10.11924/j.issn.1000-6850.casb16010020

Abstract

Abstract: An appropriate two- dimensional electrophoresis proteome analysis system of cucumber female flower’s corolla was established by optimizing the parameters including the protein extraction method, the pH gradient of IPG strip, isoelectric focusing conditions, sample application, and dyeing method. The result showed that phenol extraction method could obtain more protein with higher purity. Two- dimensional electrophoresis maps using 17 cm pH 4-7 IPG strip had no overlapping and protein points distributed evenly. Good protein points were obtained in the two-dimensional electrophoresis maps using optimized isoelectric focusing condition with added low-pressure desalination step and time. When the sample application increased to 300 μg, the number of protein points reached 400 and the low abundance protein points were also clear. More protein points were obtained in the two-dimensional electrophoresis maps using silver staining than colloid Coomassie brilliant blue staining. This study obtained clear background, high resolution, and high quality two-dimensional electrophoresis maps with protein points distributed evenly. It laid a foundation for the research on cucumber female flower’s corolla proteomics.

CLC Number:

S642.2

References

[1] 朱玉贤,李毅,郑晓峰. 现代分子生物学[M].北京.高等教育出版社,2007.
[2] 路铁刚,丁毅.分子遗传学[M]. 北京.高等教育出版社,2008.
[3] 范海延,陈捷,张春宇,等. 适于黄瓜叶片蛋白质组分析的双向电泳方法最佳条件的研究[J]. 沈阳农业大学学报,2008,39(3):365-367.
[4] 黄雅芳,杨瑞,王志忠,等. 薄皮甜瓜果实总蛋白质双向电泳优化体系的建立[J]. 中国农学通报,2014,30(19):98-104.
[5] 黎飞,徐秋芳,臧宪朋,等. 番茄子叶总蛋白双向电泳体系的建立[J]. 园艺学报,2010,37(4):661-668.
[6] 赵玲玲,毕青,冀瑞琴,等. 大白菜根系蛋白质双向电泳体系优化[J]. 分子植物育种,2015,13(1):178-183.
[7] Natarajan S, Xu C, Garrett W M, et al. Comparison of protein solubilization methods suitable for proteomic analysis of soybean seed proteins[J]. Anal Biochem,2005,342(2):214-220.
[8] Wu F S, M Y Wang. Extraction of proteins for sodium dodecyl sulfate-polyacrylamide gel electrophoresis from protease-rich plant tissues[J]. Analytical biochemistry,1984,139(1):100-103.
[9] 张侨,韩莹琰,范双喜,等. 叶用莴苣叶片蛋白质双向电泳技术体系的建立[J]. 中国农学通报,2010,26(13):279-283.
[10] 王珊珊,郝宇涵,范海延,等. 黄瓜根系蛋白质组学分析的双向电泳技术条件优化[J]. 沈阳农业大学学报,2012,43(2):233-236.
[11] 张洁,孙一凡,张荻,等. 百子莲茎尖分生组织蛋白质双向电泳体系的建立[J]. 上海交通大学(农业科学版),2015,33(3):43-52.
[12] Carpentier S C, Witters E, Laukens K, et al. Preparation of protein extracts from recalcitrant plant tissues: An evaluation of different methods for two dimensional gel electrophoresis analysis[J]. Proteomics,2005,5:2497-2507.
[13] 王新芳,郭娜,郭祥龙,等. 萌发期大豆总蛋白提取及双向电泳体系的优化[J]. 中国油料作物学报,2015,37(2):220-226.
[14] 蒋林惠,姜丽,徐银,等. 甜椒果实总蛋白质双向电泳优化体系的建立[J]. 食品工业科技,2015,36(4):84-88.
[15] 阚雪芹,方向明,刘文倩,等. 大丽菊花瓣衰老期差异表达蛋白质组的分析[J]. 江苏农业学报,2011,27(2):455-457.
[16] 周雪,冯辉,冀瑞琴. 适合双向电泳的大白菜花蕾蛋白质提取及浓度测定方法[J]. 分子植物育种,2013,11(2):249-254.
闫伟丽,钟俐,王贤磊,等. 甜瓜叶片蛋白质的提取和双向电泳体系的优化[J]. 中国农学通报,2014,30(19):105-110.
[17] 刘倩,阮松林,俞晓平,等. 适于茭白茎部蛋白质组分析的双向电泳技术体系的建立[J]. 浙江农业学报,2010,22(3):281-286.
[18] 任丽萍,范海延,王珊珊,等. 黄瓜叶片蛋白质双向电泳样品分级优化[J]. 西北植物学报,2011,31(8):1706-1710.
[19] 任燕,陈暄,黎星辉,等. 茶树雌蕊总蛋白质双向电泳分离体系的建立[J]. 茶树科学,2013,33(5):420-428.
[20] 林梓浩,李玉红,董从娟,等. 黄瓜叶片细胞壁蛋白双向电泳体系的建立[J]. 西北农林科技大学学报(自然科学版),2013,41(9):91-104.
[21] 董从娟,李玉红,林梓浩,等. 黄瓜叶片胞间隙蛋白质双向电泳体系的建立[J]. 西北植物学报,2013,33(1):0183-0189.
[22] 王珊珊,郝宇涵,范海延,等. 黄瓜根系蛋白质组学分析的双向电泳技术条件优化[J]. 沈阳农业大学学报,2012,43(2):233-236.
[23] 张鹏. 黄瓜果实弯曲性QTL定位及蛋白质组差异研究[D]. 哈尔滨:东北农业大学,2009.
[24] 李凤玉,潘德灼,陈伟. 黄瓜子叶节的蛋白质组双向电泳条件的优化[J]. 植物生理学通讯,2008,44(5):963-967.
[25] 郝雨涵,范海延,曲波,等. 黄瓜悬浮细胞蛋白质组双向电泳技术分析[J]. 西北植物学报,2012,32(4):0823-0828.
[26] Thiellement H, Zivy M, Damerval C, et al．Plant Proteomics: Methods and Protocols[M]. Totowa: Humana Press,2007
[27] 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.
[28] 谷瑞升,刘群录,陈雪梅,等. 一种省时高效的木本植物蛋白双向电泳分析方法[J]. 北京林业大学学报,1999,21(5):7-10.
[29] Bradford M M．A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding[J]. Analytical Biochemistry,1976,72: 248-254.
[30] 王立,张硕,侯喜林,等. 不结球白菜雌蕊蛋白质双向电泳技术体系的建立[J]. 南京农业大学学报,2012,35(1):14-20.
[31] 饶维桥,肖伟敏,张继远,等. 改良的蛋白质电泳考马斯亮蓝染色方法研究[J]. 生物技术通报,2014,9:59-64.
[33] 焦竹青,徐培磊,王振兴,等. 山葡萄花序蛋白质双向电泳技术体系的建立[J]. 果树学报,2012,29(5):945-951.
[34] 黄榕辉,陈品品,王平,等. 荔枝花蕊蛋白质双向电泳体系的建立与优化[J]. 热带作物学报,2012,33(10):1812-1818.
[35] 刘萍,邵晓琪,丁义峰,等. 不同芍药品种花瓣中营养成分含量的比较[J]. 北方园艺,2014(01):151-153.
[36] 尤垂淮,唐莉娜,王海斌,等. 烟草叶片蛋白质双向电泳技术体系的建立[J]. 中国烟草学报,2012,18(5):96-102.
[37] 郑蕊,岳思君,喻德跃,等. 枸杞花药蛋白质组双向电泳体系的建立与应用[J]. 西北植物学报,2011,31(12):2443-2448.
[38] 罗海波,陈伟,周静峰,等. 茭白总蛋白质双向电泳技术体系的建立[J]. 食品与生物技术学报,2013,32(6):581-585.
[39] 马洪雨,王占奎,俞阗,等. 适用于黄麻根部蛋白质组学分析的双向电泳技术[J]. 西北植物学报,2010,30(1):0195-0202.
[40] 陈蕊红,张改生,刘卫,等. 小麦花药蛋白质组双向电泳技术体系的优化[J]. 核农学报,2008,22(4):404-409.
[41] 郑天慧,宋波,刘珊珊,等. 大豆花瓣蛋白质双向电泳分析技术体系的优化[J]. 哈尔滨师范大学自然科学学报,2010,26(6):75-80.