Bäumlein H, Boerjan W, Nagy I, Panitz R, Inzé D, Wobus U
Zentralinstitut für Genetik und Kulturpflanzenforschung, Akademie der Wisssenschaften, Gatersleben, Federal Republic of Germany.
Mol Gen Genet. 1991 Jan;225(1):121-8. doi: 10.1007/BF00282650.
We have previously isolated a legumin gene LeB4 from Vicia faba and shown that a 4.7 kb DNA fragment containing the gene leads to seed-specific expression in transgenic tobacco plants. Here we report that the 2.4 kb upstream sequence alone, when fused to either the neomycin phosphotransferase II (nptII) gene or the beta-glucuronidase (uidA) gene, leads to high enzyme levels in transgenic seeds of both tobacco and Arabidopsis. beta-Glucuronidase (GUS) activity is especially intense in the cotyledons fading out towards the embryonal root tip, a result confirmed by in situ hybridization. Staining of endosperm cells is consistent in both species. Analysis of a series of promoter deletion mutants fused to the nptII gene and introduced into tobacco plants revealed that about 1 kb of 5'-flanking sequence is sufficient for high-level expression but indirect evidence suggests the presence of weak positive regulatory elements further upstream. Deletions leaving only 0.2 kb of upstream sequence reduce enzyme levels to less than 10%. A deletion which destroys the legumin box with its seed protein gene-specific CATGCATG motif has no obvious effects on expression levels.
我们之前从蚕豆中分离出一个豆球蛋白基因LeB4，并表明包含该基因的4.7 kb DNA片段在转基因烟草植株中可导致种子特异性表达。在此我们报告，单独的2.4 kb上游序列，当与新霉素磷酸转移酶II（nptII）基因或β-葡萄糖醛酸酶（uidA）基因融合时，在烟草和拟南芥的转基因种子中均能导致高酶水平。β-葡萄糖醛酸酶（GUS）活性在朝着胚根尖端逐渐褪色的子叶中尤为强烈，原位杂交证实了这一结果。两种植物中胚乳细胞的染色情况一致。对一系列与nptII基因融合并导入烟草植株的启动子缺失突变体的分析表明，约1 kb的5'侧翼序列足以实现高水平表达，但间接证据表明更上游存在弱的正调控元件。仅保留0.2 kb上游序列的缺失会使酶水平降低至10%以下。破坏具有种子蛋白基因特异性CATGCATG基序的豆球蛋白盒的缺失对表达水平没有明显影响。
