草地学报

›› 2000, Vol. 8 ›› Issue (2): 126-131.DOI: 10.11733/j.issn.1007-0435.2000.02.008

• 研究论文 • 上一篇    下一篇

顿河红豆草浓缩单宁生物合成相关蛋白鉴定

王玉萍, 杨茁萌, 张博   

  1. 新疆农业大学畜牧学院草原系, 乌鲁木齐, 830052
  • 收稿日期:1999-11-25 修回日期:2000-02-17 出版日期:2000-05-15 发布日期:2000-05-15

Identification of Protein Related to Biosynthesis of Condensed Tannin in Onobrychis tanaitica Spreng.

Wang Yuping, Yang Zhuomeng, Zhang Bo   

  1. Department of Grassland, Xinjiang Agicultural University, Urumq 830052
  • Received:1999-11-25 Revised:2000-02-17 Online:2000-05-15 Published:2000-05-15

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摘要： 以二倍体顿河红豆草为实验材料,运用组织培养法和香草醛-盐酸法筛选优良浓缩单宁高表达的细胞系。用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)技术,对同一细胞系不含浓缩单宁(CT-)愈伤组织和含有浓缩单宁(CT+)愈伤组织蛋白质进行鉴定。用制备型SDS-双向电泳对目标蛋白进行纯度分析,以及用SDS-PAGE法和IEF(等电聚焦)法测定该目标蛋白质分子量和等电点。最后经电印渍技术,将目标蛋白质转移至固相膜上进行氮端氨基酸序列分析。结果表明,筛先出10个优良浓缩单宁合成细胞系和相同基因型愈伤组织增殖细胞系。同一细胞系含有浓缩单宁愈伤组织比不含者多一条蛋白带,该蛋白分子量为28kD,等电点为5.7。该电印渍法将蛋白印渍至PVDF(聚偏二氟乙烯)膜上的效率较高。并测定了氮端部分氨基酸序列。

关键词: 顿河红豆草, SDS-双向电泳, 蛋白质, 浓缩单宁, 氮端氨基酸序列

Abstract: Onobrychis tanaitica (diploid) was used as material to identify protein related to biosynthesis of condensed tannin.Better cell lines of calli which express best condensed tannin were screened by means of tissure culture and Vanillin HCl.The calli of the same cell line containing condensed tannin and uncontaining condensed tannin were identified through SDS PAGE.The protein aim was purified and analysized with preparative two dimensional gel electrophoresis,the molecular weight and isoelectric point were checked by SDS PAGE and isoelectric focusing(IFE)respectively.The protein was electric transferred to solid phase membrance and the sequence of N terminal amino acids were analysized. The results showed:ten better cell lines of calli synthesis and ten same cell lines of calli proliferation were screened.There was one more special protein in calli containing condensed tannin than the calli uncontaining CT derived from the same cell line,the molecular weight and isoelectric point of the protein is 28kD and 5.7 respectively. The method of electric blotting has more efficiency. The sequence of some amino acids at N terminal was analysized.

Key words: Onobrychis tanaitica, SDS two dimensional gel electrophresis, Protein, Condensed tannin, N terminal amino acid sequence

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