Department of Molecular Biology, University of Wyoming, Laramie, Wyoming 82071, USA.
Plant Physiol. 2012 Aug;159(4):1309-18. doi: 10.1104/pp.112.199737. Epub 2012 Jun 15.
Maize (Zea mays) transformation routinely produces stable transgenic lines essential for functional genomics; however, transient expression of target proteins in maize cells is not yet routine. Such techniques are critical for rapid testing of transgene constructs and for experimental studies. Here, we report bombardment methods that depend on leaf developmental stage and result in successful expression with broad applications. Fluorescent marker genes were constructed and bombarded into five developmental regions in a growing maize leaf. Expression efficiency was highest in the basal-most 3 cm above the ligule of an approximately 50-cm growing adult leaf. Straightforward dissection procedures provide access to the receptive leaf regions, increasing efficiency from less than one transformant per cm(2) to over 21 transformants per cm(2). Successful expression was routine for proteins from full genomic sequences driven by native regulatory regions and from complementary DNA sequences driven by the constitutive maize polyubiquitin promoter and a heterologous terminator. Four tested fusion proteins, maize PROTEIN DISULFIDE ISOMERASE-Yellow Fluorescent Protein, GLOSSY8a-monomeric Red Fluorescent Protein and maize XYLOSYLTRANSFERASE, and maize Rho-of-Plants7-monomeric Teal Fluorescent Protein, localized as predicted in the endoplasmic reticulum, Golgi, and plasma membrane, respectively. Localization patterns were similar between transient and stable modes of expression, and cotransformation was equally successful. Coexpression was also demonstrated by transiently transforming cells in a stable line expressing a second marker protein, thus increasing the utility of a single stable transformant. Given the ease of dissection procedures, this method replaces heterologous expression assays with a more direct, native, and informative system, and the techniques will be useful for localization, colocalization, and functional studies.
玉米（Zea mays）转化常规产生稳定的转基因系对于功能基因组学至关重要；然而，目标蛋白在玉米细胞中的瞬时表达尚未常规化。这些技术对于快速测试转基因构建体和实验研究至关重要。在这里，我们报告了依赖叶片发育阶段的轰击方法，这些方法导致了广泛应用的成功表达。荧光标记基因被构建并轰击到一个生长中的玉米叶片的五个发育区域。在约 50 厘米长的成年叶片的近叶舌处的最基部 3 厘米处，表达效率最高。简单的解剖程序可提供可接受的叶片区域，使效率从每平方厘米不到一个转化体增加到每平方厘米 21 个以上。从天然调控区驱动的完整基因组序列和从组成型玉米多聚泛素启动子和异源终止子驱动的 cDNA 序列驱动的蛋白质的成功表达是常规的。四种测试的融合蛋白，玉米蛋白二硫键异构酶-黄色荧光蛋白、GLOSSY8a-单体红色荧光蛋白和玉米木聚糖转移酶以及玉米 Rho-of-Plants7-单体青绿色荧光蛋白，分别在预测的内质网、高尔基体和质膜中定位。瞬时和稳定表达模式之间的定位模式相似，共转化同样成功。通过瞬时转化稳定表达第二种标记蛋白的细胞也证明了共表达，从而增加了单个稳定转化体的实用性。鉴于解剖程序的简便性，这种方法用更直接、天然和信息丰富的系统替代了异源表达测定，该技术将有助于定位、共定位和功能研究。
