Isolation and Characterization of MADS-Box Genes from ARABIDOPSIS

在过去的几年中，对控制花发育的分子和遗传机制的理解取得了快速进展。这些研究已经确定了确定花分生组织身份的早期作用基因，以及决定花器官原基命运的后期作用基因。一个在花发育中起主要作用的基因家族是MADS-box基因，它编码推定的转录因子。事实上，拟南芥MADS-box基因APETALA1、CAULIFLOWER、AGAMOUS、APETALA3和pisttillata在决定花的分生组织和器官身份方面的作用已经被鉴定出来。此外，MADS-box基因在多种生物（包括人类、果蝇和酵母）中调节基因表达的作用已被证实。本提案的重点是进一步表征最近发表的3个MADS-box基因（AGL4-6），最近分离的9个基因（AGL8-9和AGL11-17），以及在拟南芥中分离和表征该重要基因家族的其他成员。为了鉴定新的MADS-box基因，将使用PCR扩增和低严格杂交。全长cDNA和基因组克隆将被分离和测序，推断出的蛋白质产物将与已经确定的蛋白质产物进行比较，以观察进化关系。RFLP定位将确定克隆基因是否与先前确定的突变接近。野生型和突变型植物的RNA印迹和原位杂交将确定基因表达的时空格局，并为这些基因与其他遗传位点之间的调控相互作用提供见解。表达正义和反义rna的转基因植物将通过农杆菌介导的转化产生，然后进行靶向的EMS突变体筛选，以确定克隆位点的候选突变。这些实验的初步结果支持了MADS-box基因在植物发育过程中发挥重要和多样作用的假设。MADS-box基因的鉴定将有助于深入了解这一重要基因家族在拟南芥中的作用和进化过程，并有助于我们了解远亲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.