Identification of Targets of Apetala3 and Pistillata Floral Homeotic Gene Action

0212222Irish, v.f.花器官是由花决定的分生组织的侧边生长而来，并以不同的身份分化。这些器官如萼片、花瓣、雄蕊或心皮的形成取决于几个花同源基因的作用，这些基因在这些发育途径中起着主要的调节作用。这两个基因aptala3 （AP3）和PISTILLATA （PI）编码MADS结构域的转录因子，调控拟南芥花瓣和雄蕊的特性。今后的工作将集中在确定花瓣和雄蕊发育相关的其他基因上，重点是确定AP3和PI调控的几个候选靶基因。对这些基因的分析可以作为解剖遗传等级的模型，通过遗传等级，植物发育过程中特定的器官和组织类型被指定。为了确定一组由这些转录因子在体内调节的靶基因，Irish博士提出了三种互补的方法。为了确定在AP3和PI作用下上调或下调的基因，将对不同突变背景下拟南芥est的表达进行微阵列分析。这应该定义了一组合理高表达的花瓣和/或雄蕊特异性基因，这些基因直接或间接地由这些花同源基因产物调节。其次，染色质免疫沉淀法将用于鉴定体内被AP3/PI蛋白结合的DNA序列。通过这种方法鉴定的序列是AP3/PI调控的直接靶点。第三，将采用功能获得遗传方法，使用激活标记，突变鉴定参与花发育途径的基因。这种方法的优势在于，它可以恢复以冗余方式起作用的下游目标基因，这将阻止传统的前向基因筛选恢复它们。通过这三种方法，可能会恢复数百个候选靶基因。这些方法并不意味着是详尽的，因为很可能只有丰富表达的基因才有可能被恢复。爱尔兰博士计划最初将进一步分析的重点放在有限数量的候选样本上，重点放在那些通过多种方法恢复的样本上。这些基因将被检测以确定它们的转录是否直接依赖于AP3/PI功能。这将确定一套直接目标，这些目标反过来对开始评估由AP3/PI控制的发展过程的范围很有价值。反过来，这些分析将阐明花同源基因产物是否在长调控级联的顶端起作用，或者这些基因产物是否直接作用于负责形态发生各个方面的一系列下游目标。描述这些主要调控基因如何影响它们的功能对于详细了解器官发生和形态发生是至关重要的，并且应该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.