Krishnan HB, Jiang G, Krishnan AH, Wiebold WJ
USDA-ARS, Plant Genetics Research Unit, 108W Curtis Hall, University of Missouri, 65211, Columbia, MO, USA
Plant Sci. 2000 Aug 22;157(2):191-199. doi: 10.1016/s0168-9452(00)00283-1.
A 53 kDa protein, which accumulates at low levels in non-nodulating Clark soybeans, was purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Polyclonal antibodies were raised against the gel-purified protein. N-terminal sequence analysis identified the 53 kDa protein as the beta-subunit of beta-conglycinin. Results from Western blot analysis, using antibodies raised against the purified beta-subunit of beta-conglycinin, revealed that accumulation of this protein was enhanced in non-nodulating soybeans when the plants were supplemented with nitrogen. Results from Northern blot analysis indicate that non-nodulating soybeans in general had lower levels of mRNA for the major soybean seed proteins. A one-time application of nitrogen to non-nodulating soybeans enhanced the accumulation of the 1.6 kb beta-conglycinin beta-subunit mRNA. The mRNA levels of the 2.1 kb beta-conglycinin alpha'-subunit and the 2.2 kb G4 glycinin in the non-nodulating soybeans were several-fold lower than in nodulating soybeans. Nitrogen application had no effect on the abundance of these RNA transcripts. The amount of RNA encoding an 8.5 kDa sulfur-rich protein was two-fold higher in non-nodulating soybeans when compared with nodulated soybeans. Nitrogen application reduced the abundance of this transcript to levels comparable with those of nodulated soybeans. Despite lower levels of the beta-subunit of beta-conglycinin, the methionine content of the total seed protein fraction was lower than that of nodulated soybeans. In contrast, non-nodulating soybeans contained more cysteine than nodulating soybeans.
一种在非结瘤克拉克大豆中低水平积累的53 kDa蛋白质，通过制备性十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）进行了纯化。针对凝胶纯化的蛋白质制备了多克隆抗体。N端序列分析确定该53 kDa蛋白质为β-伴大豆球蛋白的β亚基。使用针对纯化的β-伴大豆球蛋白β亚基产生的抗体进行的蛋白质印迹分析结果表明，当给植物补充氮时，这种蛋白质在非结瘤大豆中的积累会增强。Northern印迹分析结果表明，一般来说，非结瘤大豆中主要大豆种子蛋白的mRNA水平较低。一次性给非结瘤大豆施氮可增强1.6 kbβ-伴大豆球蛋白β亚基mRNA的积累。非结瘤大豆中2.1 kbβ-伴大豆球蛋白α'-亚基和2.2 kb G4大豆球蛋白的mRNA水平比结瘤大豆低几倍。施氮对这些RNA转录本的丰度没有影响。与结瘤大豆相比，非结瘤大豆中编码8.5 kDa富含硫蛋白的RNA量高出两倍。施氮使该转录本的丰度降低到与结瘤大豆相当的水平。尽管β-伴大豆球蛋白β亚基水平较低，但总种子蛋白组分的蛋氨酸含量低于结瘤大豆。相比之下，非结瘤大豆含有的半胱氨酸比结瘤大豆多。
