Vodkin L O, Raikhel N V
Plant Molecular Genetics Laboratory, Agricultural Research Service, United States Department of Agriculture, Beltsville, Maryland 20705.
Plant Physiol. 1986 Jun;81(2):558-65. doi: 10.1104/pp.81.2.558.
The localizations of soybean lectin (SBL) and antigenically related proteins in cotyledons and roots of lectin positive (Le(+)) and lectin negative (Le(-)) soybean cultivars were compared by light level immunocytochemistry using antibodies produced against the 120 kilodalton (kD) native seed lectin tetramer or its subunits. Lectin is present in the protein bodies of cotyledons cells as are two other seed proteins, the Kunitz trypsin inhibitor and the storage protein glycinin. Analysis of single seed extracts by immunoblotting of sodium dodecyl sulfate-polyacrylamide gels using the same antibodies, reveals up to 4 milligrams of the 30 kD seed lectin protein is present per seed in the Le(+) varieties. There is no detectable lectin in the protein bodies of Le(-) cotyledons as determined by immunocytochemistry and immunoblotting. Enzyme-linked immunosorbent assay confirmed this result to a sensitivity of less than 20 nanograms per seed. In contrast, the roots of both Le(+) and Le(-) plants bind the seed lectin antibody during immunocytochemistry, with fluorescence mainly localized in vacuole-like bodies in the epidermis. Root extracts contain a 33 kD polypeptide that binds anti-SBL antibody at an estimated minimal level of 20 nanograms per 4-day seedling, or 2.0 nanograms per primary root tip. This polypeptide is also present in the embryo axis and in leaves. The latter also contain a 26 kD species that binds seed lectin antibody. The 30 kD seed lectin subunit, however, is not detectable in roots or leaves.
通过使用针对120千道尔顿(kD)天然种子凝集素四聚体或其亚基产生的抗体,采用光镜免疫细胞化学方法,比较了凝集素阳性(Le(+))和凝集素阴性(Le(-))大豆品种子叶和根中大豆凝集素(SBL)及抗原相关蛋白的定位。凝集素与另外两种种子蛋白,即库尼茨胰蛋白酶抑制剂和贮藏蛋白大豆球蛋白一样,存在于子叶细胞的蛋白体中。使用相同抗体对十二烷基硫酸钠-聚丙烯酰胺凝胶进行免疫印迹分析单粒种子提取物,结果显示在Le(+)品种中,每粒种子中存在高达4毫克的30 kD种子凝集素蛋白。通过免疫细胞化学和免疫印迹法测定,Le(-)子叶的蛋白体中未检测到凝集素。酶联免疫吸附测定证实了这一结果,其灵敏度低于每粒种子20纳克。相比之下,在免疫细胞化学过程中,Le(+)和Le(-)植株的根均能结合种子凝集素抗体,荧光主要定位于表皮中类似液泡的小体。根提取物含有一种33 kD的多肽,每4日龄幼苗中该多肽与抗SBL抗体结合的估计最低水平为20纳克,或每根尖为2.0纳克。这种多肽也存在于胚轴和叶片中。叶片中还含有一种26 kD的物质,它能结合种子凝集素抗体。然而,在根或叶中未检测到30 kD的种子凝集素亚基。