TRANSGENE EXPRESSION VARIABILITY (POSITION EFFECT) OF CAT AND GUS REPORTER GENES DRIVEN BY LINKED DIVERGENT T-DNA PROMOTERS
TRANSGENE EXPRESSION VARIABILITY (POSITION EFFECT) OF CAT AND GUS REPORTER GENES DRIVEN BY LINKED DIVERGENT T-DNA PROMOTERS
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DOI：
10.1007/bf00036805
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发表时间：
1991-07-01
影响因子：
5.1
通讯作者：
VELTEN, J
中科院分区：
文献类型：
--
作者：
PEACH, C;VELTEN, J
同时测定了45个单独转化的克隆烟草愈伤组织系的氯霉素乙酰转移酶（CAT）和β-葡萄糖醛酸酶（GUS）活性，所述活性是由相邻的和不同的甘露碱（mas）启动子驱动的引入的报告基因的表达产生的。排除其中一种或两种酶活性基本为零的品系，报告基因的活性在个体转化体之间变化多达136（CAT）和175（GUS）的因子。在高度的克隆间表达变异性之上叠加的是3-4倍的克隆内变异性。由于甘露碱启动子的调控特性，观察到的克隆内报告基因活性程度可能更极端，但考虑到基于报告基因的转基因功能和结构分析的局限性，仍必须加以解决。有没有引入CAT和GUS基因的表达水平之间的一致的相关性，因为在个别行内的GUS CAT活性（nmol min-1 mg-1）的比例从0.05到49不等。甚至来自两个直接相邻的启动子区域（均包含在479 bp TR-DNA片段内）的趋异转录也不足以保证两个连接的转基因的同时表达。将我们的定量数据与已发表的转基因表达变异性数据进行比较，以检查个体转化体中表达水平的总体分布。所得频率分布表明，大多数转化体以相对低的水平表达引入的转基因，这表明潜在大量的农杆菌介导的转化事件可能导致沉默转基因。
Forty-five individually transformed clonal tobacco callus lines were simultaneously assayed for both chloramphenicol acetyltransferase (CAT) and beta-glucuronidase (GUS) activity resulting from expression of introduced reporter genes driven by the adjacent and divergent mannopine (mas) promoters. Excluding lines in which one or both of the enzyme activities was essentially zero, the activities of the reporter genes varied by as much as a factor of 136 (CAT) and 175 (GUS) between individual transformants. Superimposed upon the high degree of inter-clonal expression variability was an intra-clonal variability of 3-4-fold. The observed degree of intra-clonal reporter gene activity may be more extreme because of the regulatory characteristics of the mannopine promoters, but must still be addressed when considering the limitations of reporter gene-based analysis of transgene function and structure. There was no consistent correlation between the expression levels of the introduced CAT and GUS genes since the ratio of GUS to CAT activities (nmol min-1 mg-1) within individual lines varied from 0.05 to 49. Even divergent transcription from two directly adjacent promoter regions (both contained within a 479 bp TR-DNA fragment) is insufficient to guarantee concurrent expression of two linked transgenes. Our quantitative data were compared to published data of transgene expression variability to examine the overall distribution of expression levels in individual transformants. The resulting frequency distribution indicates that most transformants express introduced transgenes at relatively low levels, suggesting that a potentially large number of Agrobacterium-mediated transformation events may result in silent transgenes.