Breyne P, Gheysen G, Jacobs A, Van Montagu M, Depicker A
Laboratorium voor Genetica, Universiteit Gent, Belgium.
Mol Gen Genet. 1992 Nov;235(2-3):389-96. doi: 10.1007/BF00279385.
T-DNA vectors were constructed which carry a beta-glucuronidase (gusA) gene fused to the promoter of the nopaline synthase (nos) gene and the 3' end of the octopine synthase (ocs) gene. This reporter gene was cloned at different locations and orientations towards the right T-DNA border. For each construct, between 30 and 60 stably transformed calli were analysed for beta-glucuronidase activity. Depending on the T-DNA configuration, distinct populations of gusA-expressing calli were obtained. Placing the reporter gene in the middle of the T-DNA results in relatively low expression levels and a limited inter-transformant variability. Placing the gene with its promoter next to the right border led to an increase in both the mean activity and the variability level. With this construct, some of the calli expressed the gusA gene at levels four to five times higher than the mean. In all these series, at least 30% of the calli contained reporter gene activities that were less than half of the mean expression level. Separating the gusA gene from the right T-DNA border by an additional 3'-untranslated region, derived from the nos gene, resulted in an increase in the mean expression to a level almost four times higher than that of constructions carrying the reporter gene in the middle of the T-DNA. Moreover, the number of transformants with extremely low activities decreased by at least 50% and this resulted in significantly lower inter-transformant variability independently of the orientation of the reporter gene on the T-DNA.
构建了携带与胭脂碱合成酶(nos)基因启动子和章鱼碱合成酶(ocs)基因3'末端融合的β-葡萄糖醛酸酶(gusA)基因的T-DNA载体。该报告基因被克隆到不同位置,并朝着T-DNA右边界以不同方向排列。对于每个构建体,分析了30至60个稳定转化的愈伤组织的β-葡萄糖醛酸酶活性。根据T-DNA的构型,获得了不同的gusA表达愈伤组织群体。将报告基因置于T-DNA中间会导致相对较低的表达水平和有限的转化体间变异性。将带有启动子的基因置于右边界旁边会导致平均活性和变异性水平都增加。使用这种构建体,一些愈伤组织中gusA基因的表达水平比平均值高四到五倍。在所有这些系列中,至少30%的愈伤组织所含报告基因活性低于平均表达水平的一半。通过来自nos基因的额外3'非翻译区将gusA基因与T-DNA右边界分开,导致平均表达增加到几乎比将报告基因置于T-DNA中间的构建体高四倍的水平。此外,活性极低的转化体数量减少了至少50%,这导致转化体间变异性显著降低,而与报告基因在T-DNA上的方向无关。