Studies of function and transcriptional regulation of auxin-induced genes

我们通过对生长素调控基因arcA、parB和parC的功能和表达调控的研究，在分子水平上深入了解生长素的作用机制。从烟草by -2细胞中分离得到的arcA基因在应用生长素诱导细胞分裂之前开始表达。序列分析显示，arcA属于一个扩展的基因家族，包括G蛋白β亚基的基因。这些蛋白质都有一系列由40个氨基酸组成的重复序列，称为WD-40重复序列。WD-40重复序列家族的成员参与多种细胞功能。大多数已知的WD-40蛋白形成多蛋白复合物，有时通过WD-40重复区与其他蛋白相互作用。因此，我们利用双杂交筛选技术在酵母中分离了多个arcA相互作用蛋白的cDNA克隆，并通过体外系统检测了它们与arcA产物的直接结合能力。其中一个cDNA克隆与哺乳动物细胞中电压依赖性K^+通道的β亚基具有广泛的同源性。针对arcA产物的抗体显示，arcA产物在BY-2传代培养后2天达到最高水平，此时细胞分裂最活跃。以GUS为报告基因，对烟草叶肉原生质体中parA、parB和parC的生长素响应顺式元件进行了研究。包括点突变实验在内的详细分析表明，生长素响应性需要不同位置序列的相互作用。此外，不同的核蛋白结合到这些基因的生长素响应元件上。虽然parA、parB和parC都是从烟草叶肉原生质体中分离出来的，但生长素介导的转录激活依赖于不同的顺式和反式因子。这些结果表明，生长素的多种生理作用是由转录调控的复合分子机制所支持的。

We have studied about functions and modulation of expression of auxin-regulated genes, arcA,parB and parC to gain insight into the action mechanism of auxins at the molecular level. The initiation of expression of arcA,isolated from tobacco BY-2 cells, by the application of auxin precedes the induction of cell division. Sequence analysis revealed that arcA belongs to an expanding gene family, including the genes for beta subunits of G proteins. These proteins all have a series of internal repeats of 40 amino acids called the WD-40 repeat. Members of the WD-40 repeat family are involved in various cellular functions. Most known WD-40 proteins form multiprotein complexes, sometimes interacting with other proteins through the WD-40 repeat region. So we isolated several cDNA clones of arcA-interacting proteins using two-hybrid screen in yeasts and examined their direct binding capabilities with arcA products by the in vitro system. One of these cDNA clones had the extensive homology to a beta subunit of voltage-dependent K^+ channel well characterized in mammalian cells. The antibodies against arcA products revealed that arcA products accumulated at the maximum level 2 days after the subculture of BY-2, at which time the cells were most actively dividing.We investigated auxin-responsive cis elements of parA,parB and parC from tobacco mesophyll protoplasts in transgenic tobaccos using GUS as a reporter gene. Detailed analysis including point mutation experiments showed that the interaction of separately located sequences was required for the auxin-responsiveness. Furthermore, different nuclear proteins bound to each of auxin-responsive elements of these genes. Although parA,parB and parC are all isolated from tobacco mesophyll protoplasts, auxin mediated activation of transcription depends on different cis and trans factors. These results suggest that various physiological effects of auxins are underlain by the composite molecular mechanisms of trascriptional regulation.