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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 1 0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 2 8k D,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
The diploid Donax japonicus was used as experimental material, and the tissue culture method and vanillin-hydrochloric acid method were used to screen the highly concentrated tannin highly expressed cell lines. The CT-callus and CT + -containing callus proteins of the same cell line were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) . The purity of the target protein was analyzed by preparative SDS-two-dimensional electrophoresis, and the molecular weight and isoelectric point of the target protein were determined by SDS-PAGE and IEF (isoelectric focusing). Finally, by electroblotting technique, the target protein was transferred to the solid phase membrane for N-terminal amino acid sequence analysis. The results showed that 10 excellent tannin-producing cell lines and the same genotype callus proliferating cell line were screened out first. The same cell line contains concentrated tannin callus more than a non-containing protein band, the molecular weight of 28k D, isoelectric point of 5.7. The electroblotting method is more efficient for printing proteins onto PVDF (polyvinylidene difluoride) films. The amino acid sequence of the nitrogen end was determined