Identification of Targets of Apetala3 and Pistillata Floral Homeotic Gene Action

0212222 Irish，V.F.花器官从花决定的分生组织中侧向生长，并以不同的身份分化。 这些器官的规格为萼片，花瓣，雄蕊或心皮取决于几个花的同源异型基因的行动，作为这些发育途径的主调节器。 两个这样的基因，APETALA 3（AP 3）和PISTILLATA（PI），编码含有MADS结构域的转录因子，调节拟南芥中花瓣和雄蕊身份的特化。拟议的工作将集中在确定参与花瓣和雄蕊发育的其他基因，特别强调确定由AP 3和PI调控的几个候选靶基因。这些基因的分析可以作为一个模型，解剖的遗传层次，特定的器官和组织类型在植物发育过程中指定。为了确定一组由这些转录因子在体内调控的靶基因，Irish博士提出了三种互补的方法。 将进行微阵列分析，比较一组拟南芥EST在各种突变体背景中的表达，以确定那些响应于AP 3和PI作用而上调或下调的基因。 这应该定义一组合理的高度表达的花瓣和/或雄蕊特异性基因，直接或间接地由这些花同源异型基因产物调控。第二，染色质免疫沉淀将被用于鉴定在体内被AP 3/PI蛋白结合的DNA序列。 通过这种方法鉴定的序列是作为AP 3/PI调节的直接靶标的良好候选者。 第三，将采用功能获得遗传学方法，使用激活标签，突变鉴定参与花发育途径的基因。 这种方法的优点在于，它可以导致以冗余方式起作用的下游靶基因的恢复，这将阻止它们通过传统的正向遗传筛选的恢复。通过这三种方法可以潜在地恢复数百个候选靶基因。 这些方法并不意味着是详尽的，因为很可能只有大量表达的基因才有可能被回收。 Irish博士计划首先对有限数量的候选人进行进一步的分析，重点是那些通过多种方法恢复的人。 将检查这些基因以确定它们的转录是否直接依赖于AP 3/PI功能。 这将确定一组直接目标，这些目标反过来对开始评估由AP 3/PI控制的发育过程的范围很有价值。 反过来，这些分析将揭示花同源异型基因产物是否在长的调控级联的顶部起作用，或者这些基因产物是否直接起作用来调控负责形态发生各个方面的下游靶点。 描述这些主调控基因如何影响其功能对于详细了解器官发生和形态发生如何发生至关重要，并应作为其他植物系统中这些过程的模型。

0212222Irish, V.F.Floral organs arise as lateral outgrowths from the florally determined meristem, and differentiate with distinct identities. The specification of these organs as sepals, petals, stamens or carpels depends on the action of several floral homeotic genes which act as master regulators of these developmental pathways. Two such genes, APETALA3 (AP3) and PISTILLATA (PI), encode MADS domain-containing transcription factors that regulate the specification of petal and stamen identity in Arabidopsis. The proposed work will focus on identifying other genes involved in petal and stamen development, with a particular emphasis on identifying several candidate target genes regulated by AP3 and PI. The analysis of such genes can serve as a model for dissecting the genetic hierarchies by which particular organs and tissue types are specified during plant development. In order to identify a set of target genes regulated in vivo by these transcription factors, Dr. Irish proposes three complementary approaches. Microarray analyses comparing the expression of a set of Arabidopsis ESTs in various mutant backgrounds will be carried out in order to define those genes that are up- or down-regulated in response to AP3 and PI action. This should define a set of reasonably highly expressed petal and/or stamen specific genes that are directly or indirectly regulated by these floral homeotic gene products. Second, chromatin immunoprecipitation will be employed to identify DNA sequences that are bound in vivo by AP3/PI proteins. Sequences identified by this approach are good candidates for being direct targets of AP3/PI regulation. Third, a gain-of-function genetic approach will be employed, using activation tagging, to mutationally identify genes that are involved in the floral developmental pathway. This approach has the advantage that it can result in the recovery of downstream target genes that act in a redundant fashion, which would preclude their recovery by traditional forward genetic screens. Potentially hundreds of candidate target genes may be recovered by these three approaches. These approaches are not meant to be exhaustive, as it is likely that only abundantly expressed genes are likely to be recovered. Dr. Irish plans to initially focus further analyses on a limited number of candidates, focussingon those recovered by more than one method. Such genes will be examined to determine whether their transcription is directly dependent on AP3/PI function. This will define a set of direct targets that will in turn be valuable in beginning to assess the spectrum of developmental processes controlled by AP3/PI. In turn these analyses will shed light on the question of whether the floral homeotic gene products act at the top of a long regulatory cascade, or whether these gene products act directly to regulate a wide array of downstream targets responsible for various aspects of morphogenesis. Characterizing how such master regulatory genes effect their functions will be crucial for a detailed understanding of how organogenesis and morphogenesis ensue, and should serve as a model for these processes in other plant systems.