Prakash N Shiva, Prasad V, Chidambram Thillai P, Cherian Shoba, Jayaprakash T L, Dasgupta Santanu, Wang Qi, Mann Michael T, Spencer T Michael, Boddupalli Raghava S
Monsanto Research Centre, #44/2A, Vasanths' Business Park, Bellary Road, NH:7, Hebbal, Bangalore, India.
Transgenic Res. 2008 Aug;17(4):695-704. doi: 10.1007/s11248-007-9149-0. Epub 2007 Oct 19.
Identification of an appropriate selection agent and its corresponding selectable marker gene is one of the first steps in establishing a transformation protocol for a given plant species. As the promoter controls expression level of the genes, the promoter driving the selectable marker gene can affect transformation. However, investigations into the direct effect of promoters driving selectable marker on transformation are lacking in the literature though many reports of relative strengths of promoters driving reporter genes like GUS or CAT or GFP are available. In the present study, we have compared rice Actin1 and CaMV.35S (commonly used promoters in monocotyledonous plant transformation) promoters driving nptII for their effectiveness in paromomycin selection of transgenic corn events. To enable statistically meaningful analysis of the results, a large sample size of nearly 5,000 immature embryos (explants) was employed producing approximately 1,250 independent events from each of the two constructs in four independent experiments. The rate of appearance of resistant calli and percentage of resistant calli recovered was higher with P-Os.Actin1/nptII/nos3' as compared to P-CaMV.35S/nptII/nos3' in all four experiments. There was no appreciable difference either in the frequency of plant regeneration or in the morphological characteristics of plants recovered from the two constructs. Although the escape rate trended lower with P-Os.Actin1 as compared to P-CaMV.35S, the recovery of low copy events was significantly higher with P-CaMV.35S. The higher transformation frequency with P-Os.Actin1 could be related to the strength of this promoter as compared to P-CaMV.35S in the explants and/or calli. Based on these results, we infer that the promoter driving the selectable marker is an important factor to be considered while establishing a high throughput transformation protocol as it could not only influence the transformation frequency but also the copy number of the transgene in the recovered transgenics.
确定合适的选择剂及其相应的选择标记基因是为特定植物物种建立转化方案的首要步骤之一。由于启动子控制基因的表达水平，驱动选择标记基因的启动子会影响转化。然而，尽管有许多关于驱动报告基因（如GUS、CAT或GFP）的启动子相对强度的报道，但文献中缺乏对驱动选择标记的启动子对转化的直接影响的研究。在本研究中，我们比较了水稻肌动蛋白1启动子（Actin1）和花椰菜花叶病毒35S启动子（CaMV.35S，单子叶植物转化中常用的启动子）驱动新霉素磷酸转移酶II基因（nptII）在巴龙霉素筛选转基因玉米事件中的有效性。为了使结果具有统计学意义的分析，我们使用了近5000个未成熟胚（外植体）的大样本量，在四个独立实验中，从两种构建体中的每一种产生了约1250个独立事件。在所有四个实验中，与P-CaMV.35S/nptII/nos3'相比，P-Os.Actin1/nptII/nos3'的抗性愈伤组织出现率和回收的抗性愈伤组织百分比更高。从两种构建体再生的植株的再生频率或形态特征没有明显差异。尽管与P-CaMV.35S相比，P-Os.Actin1的逃逸率呈下降趋势，但P-CaMV.35S的低拷贝事件回收率显著更高。与P-CaMV.35S相比，P-Os.Actin1在更高的转化频率可能与该启动子在外植体和/或愈伤组织中的强度有关。基于这些结果，我们推断，在建立高通量转化方案时，驱动选择标记的启动子是一个需要考虑的重要因素，因为它不仅会影响转化频率，还会影响回收的转基因植株中转基因的拷贝数。
