Isolation and Characterization of MADS-Box Genes from ARABIDOPSIS

在过去的几年里，在了解控制花发育的分子和遗传机制方面取得了快速的进展。这些研究已经确定了确定花分生组织特性的早期作用基因和决定花器官原基命运的后期作用基因。在花发育中起主要作用的一个基因家族是MADS-box基因，它编码可能的转录因子。事实上，5个不同的拟南芥MADS-box基因，APETALA1，花椰菜，AGAMOUS，APETALA3，和雌蕊，已经根据它们在指定花分生组织和器官特性方面的作用而被表征。此外，MADS-box基因已被证明在调节不同生物中的基因表达方面发挥作用，包括人类、果蝇和酵母。这项建议的重点是对最近发表的3个MADS-box基因(AGL4-6)和最近分离的9个新基因(AGL8-9和AGL11-17)进行进一步的鉴定，以及对这一重要基因家族中其他成员的分离和鉴定。为了鉴定新的MADS-box基因，将使用PCR扩增和低严格杂交。全长的cdna和基因组克隆将被分离和测序，推导出的蛋白质产物将与那些已经鉴定的产物进行比较，以寻找进化关系。RFLP图谱将确定克隆的基因是否位于先前发现的突变附近。野生型和突变型植物的RNA印迹和原位杂交将定义基因表达的时空模式，并为这些基因与其他遗传位点之间的调控相互作用提供深入的见解。通过农杆菌介导的转化获得表达正义和反义RNA的转基因植株，然后进行有针对性的EMS突变筛选，以确定克隆的基因座上的候选突变。这些实验的初步结果支持MADS-box基因在植物发育中发挥着重要而多样的作用的假说。MADS-box基因的特征将有助于我们深入了解这一重要基因家族在拟南芥中的作用和进化，并有助于我们理解远缘植物和其他生物中的相关基因。

Rapid progress has been made in the past few years towards understanding the molecular and genetic mechanisms controlling flower development. These studies have identified early-acting genes that specify the identity of floral meristems, and later-acting genes that determine the fate of floral organ primordia. One family of genes that has emerged as playing a major role in flower development are the MADS-box genes, which encode putative transcription factors. Indeed, five different Arabidopsis MADS-box genes, APETALA1, CAULIFLOWER, AGAMOUS, APETALA3, and PISTILLATA, have been characterized in terms of their roles in specifying both floral meristem and organ identity. Furthermore, MADS-box genes have documented roles in regulating gene expression in diverse organisms, including humans, Drosophila, and yeast. The focus of this proposal is on the further characterization of three recently published MADS-box genes (AGL4-6), on nine additional genes that were recently isolated (AGL8-9 and AGL11-17), and on the isolation and characterization of additional members of this important gene family in Arabidopsis. To identify new MADS-box genes, PCR amplifications and low stringency hybridizations will be used. Full length cDNA and genomic clones will be isolated and sequenced, and deduced protein products will be compared to those already identified to look at evolutionary relationships. RFLP mapping will determine if the cloned gene maps near a previously identified mutation. RNA blotting and in situ hybridizations in wild type and mutant plants will define the temporal and spatial pattern of gene expression, and provide insights into the regulatory interactions between these and other genetic loci. Transgenic plants that express sense and antisense RNAs will be generated by Agrobacterium-mediated transformation, followed by targeted EMS mutant screens to identify candidate mutations in cloned loci. Preliminary results from these experiments support the hypothe sis that MADS-box genes play important and diverse roles in plant development. Characterization of MADS-box genes will provide insights into the roles and evolution of this important gene family in Arabidopsis, and will contribute to our understanding of related genes in distantly related plants and other organisms.