Saalbach I, Pickardt T, Machemehl F, Saalbach G, Schieder O, Müntz K
Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany.
Mol Gen Genet. 1994 Jan;242(2):226-36. doi: 10.1007/BF00391017.
The coding region of the 2S albumin gene of Brazil nut (Bertholletia excelsa H.B.K.) was completely synthesized, placed under control of the cauliflower mosaic virus (CaMV) 35S promoter and inserted into the binary vector plasmid pGSGLUC1, thus giving rise to pGSGLUC1-2S. This was used for transformation of tobacco (Nicotiana tabacum L. cv. Petit Havanna) and of the grain legume Vicia narbonensis L., mediated by the supervirulent Agrobacterium tumefaciens strain EHA 101. Putative transformants were selected by screening for neomycin phosphotransferase (NPT II) and beta-glucuronidase (GUS) activities. Transgenic plants were grown until flowering and fruiting occurred. The presence of the foreign gene was confirmed by Southern analysis. GUS activity was found in all organs of the regenerated transgenic tobacco and legume plants, including the seeds. In the legume, the highest expression levels of the CaMV 35S promoter-controlled 2S albumin gene were observed in leaves and roots. 2S albumin was localized in the vacuoles of leaf mesophyll cells of transgenic tobacco. The Brazil nut protein was present in the 2S fraction after gel filtration chromatography of the legume seed proteins and could be clearly identified by immunoblotting. Analysis of seeds from the R2 progenies of the legume and of transgenic tobacco plants revealed Mendelian inheritance of the foreign gene. Agrobacterium rhizogenes strain RifR 15834 harbouring the binary vector pGSGLUC1-2S was also used to transform Pisum sativum L. and Vicia faba L. Hairy roots expressed the 2S albumin-specific gene. Several shoots were raised but they never completely rooted and no fertile plants were obtained from these transformants.
巴西坚果（Bertholletia excelsa H.B.K.）2S白蛋白基因的编码区被完全合成，置于花椰菜花叶病毒（CaMV）35S启动子的控制之下，并插入二元载体质粒pGSGLUC1，从而产生了pGSGLUC1 - 2S。该质粒用于在超强致病力的根癌农杆菌菌株EHA 101介导下转化烟草（Nicotiana tabacum L. cv. Petit Havanna）和豆科植物窄叶野豌豆（Vicia narbonensis L.）。通过筛选新霉素磷酸转移酶（NPT II）和β-葡萄糖醛酸酶（GUS）活性来选择推定的转化体。转基因植物生长至开花结果。通过Southern分析确认了外源基因的存在。在再生的转基因烟草和豆科植物的所有器官中都发现了GUS活性，包括种子。在豆科植物中，在叶和根中观察到CaMV 35S启动子控制的2S白蛋白基因的最高表达水平。2S白蛋白定位于转基因烟草叶肉细胞的液泡中。豆科植物种子蛋白经凝胶过滤层析后，巴西坚果蛋白存在于2S组分中，并且可以通过免疫印迹清楚地鉴定出来。对豆科植物和转基因烟草植物R2后代种子的分析表明外源基因呈孟德尔遗传。携带二元载体pGSGLUC1 - 2S的发根农杆菌菌株RifR 15834也用于转化豌豆（Pisum sativum L.）和蚕豆（Vicia faba L.）。毛状根表达2S白蛋白特异性基因。诱导出了几株芽，但它们从未完全生根，并且从这些转化体中未获得可育植株。
