Isolation of Auxin Signal Transduction Mutants in Arabidopsis

植物激素生长素参与生长发育的许多方面，但人们对这种激素如何在植物细胞内引起变化知之甚少。众所周知，当植物细胞暴露于生长素时，某些基因会被激活：没有生长素，它们就不会表达(即它们的蛋白质产物不会产生)，而有了生长素，它们就会迅速高水平表达。人们还知道，这些基因启动子(即控制基因表达时间和地点的基因部分)内的特定DNA序列是对生长素的这种反应所必需的。在生长素进入植物细胞后，参与识别生长素的分子数量未知，并导致这些基因被激活。该项目的目标是确定参与这一“生长素信号转导”过程的一些分子。这个项目中采用的遗传方法包括在模式植物拟南芥中制造突变体，这些突变体显示生长素反应基因的异常表达。为此，产生了携带由生长素反应启动子控制的工程化可选择标记基因的转基因植物。转基因植物被诱变，然后这些植物的种子在允许选择表现出可选择标记基因异常表达的植物的条件下生长。使用的可选标记之一是对抗生素潮霉素产生抗药性的基因。非突变的转基因植物被抗生素杀死，因为它们没有含有足够的生长素来开启抗性基因。然而，少数突变植株能够生长，因为抗性基因在它们身上的表达比非突变植株更高。使用的另一个可选标记是编码酒精脱氢酶(ADH)的基因。ADH将烯丙醇转化为有毒物质丙烯醛。先用生长素处理，然后用烯丙醇处理的非突变转基因植物被杀死，因为生长素处理激活了ADH基因。然而，少数突变植株存活了下来，因为ADH基因在它们体内的表达水平较低。一种假设是，这些突变系包含了与生长素反应有关的分子的变化。对于一些突变体，通过观察除了可选择的标记基因之外，其他生长素反应基因是否在突变体中显示出变化的表达来检验这一假设。该项目的一个目标是继续鉴定表现出生长素反应基因表达变化的新突变体。这些突变中的几个将被详细分析，突变基因将被克隆，以发现它们编码什么分子。这项工作对加深我们对植物激素生物学和植物信号转导过程的了解具有很大的潜力。

The plant hormone auxin is involved in many aspects of growth and development, yet little is known about exactly how this hormone causes changes within plant cells. It is known that when plant cells are exposed to auxin, certain genes are activated: without auxin, they are not expressed (i.e., their protein products are not made), and with auxin they rapidly become expressed at high levels. It is also known that specific DNA sequences within the promoters of these genes (i.e, the parts of the genes that control when and where the genes are expressed) are needed for this response to auxin. An unknown number of molecules are involved in recognizing auxin after it enters the plant cell, and causing these genes to be activated. The goal of this project is to identify some of the molecules involved in this "auxin signal transduction" process. The genetic approach taken in this project involves making mutants in the model plant Arabidopsis that show aberrant expression of auxin-responsive genes. Towards this end, transgenic plants were generated that carried engineered selectable marker genes controlled by auxin responsive promoters. The transgenic plants were mutagenized, then seeds from these plants were grown under conditions that allowed for the selection of plants that showed aberrant expression of the selectable marker genes. One of the selectable markers used was a gene that confers resistance to the antibiotic hygromycin. Non-mutant, transgenic plants were killed by the antibiotic, because they did not contain enough auxin to switch on the resistance gene. However, a few mutant plants were able to grow, because the resistance gene was expressed more highly in them than in non-mutant plants. The other selectable marker used was a gene encoding the enzyme alcohol dehydrogenase (ADH). ADH converts allyl alcohol to a toxic substance, acrolein. Non-mutant transgenic plants that were treated first with auxin, then with allyl alcohol, were killed because the ADH gene was switched on by the auxin treatment. However a few mutant plants survived the treatments because the ADH gene was expressed at lower levels in them. One hypothesis is that these mutant lines contain alterations in the molecules involved in responding to auxin. For some of the mutants, this hypothesis has been tested by seeing if other auxin responsive genes, in addition to the selectable marker genes, show altered expression in the mutants. One goal of thsi project is to continue identifying new mutants that show altered expression of auxin-responsive genes. A few of these mutants will be analyzed in detail, and the mutant genes will be cloned in order to discover what molecules they encode. This work has great potential to enhance our understanding of both plant hormone biology, and signal transduction processes in plants.