Controlled Deletional Mutagenesis and Gene Homing in Arabidopsis

在拟南芥中，缺失诱变没有像插入诱变那样广泛使用，因为大的缺失通常不通过配子传递，或者在发育早期是致命的。研究者将通过控制其染色体位置、发育时间和起源亲本对小缺失进行系统研究。 她将使用位点特异性噬菌体P1编码的Cre重组酶删除含有loxP的T-DNA供体位点和附近含有loxP的转座子之间的染色体片段，从而进行已知大小的缺失。使用已经测试的含有Cre重组酶的loxP识别位点的转座子“发射台”，她将从几个转座子供体位点中的每一个鉴定3-4个以不同距离重新插入的转座子，所述距离从几个转座子到几百万个不等，以进行这些研究。 她将使用从组成型植物启动子以及从化学诱导型启动子表达的Cre重组酶基因。 她将通过PCR和遗传学方法检测缺失（和倒位）。 她将确定缺失的遗传传递性，并分析在发芽后不同时间诱导缺失的植物的发育。这项工作将增加对缺失和缺失杂合性的有害影响的理解，并提供控制缺失的大小、染色体位置和时间的方法。第二个目标是为开发基于转座子的方法将基因靶向到其原始染色体位置奠定基础。 这项技术，研究人员称之为基因“归巢”，以区别于同源性为基础的基因靶向，将使用位点特异性重组靶向启动子报告盒转座子破坏的基因。 如果成功的话，这项工作将首次有可能在基因的原始染色质背景下研究植物基因调控序列的精确改变。 基因归巢方法将使用转座子通过用启动子-报告基因盒替换转座子上的loxP包围的标记基因来将基因靶向其原始染色质环境。她将使用现有的植物与loxP转座子和转座子发射台，以确定是否有效的“盒替换”技术在动物细胞中开发的也可以用来整合DNA片段有效地在拟南芥中的loxP位点对之间。 这项工作将建立一个由被破坏的基因的启动子驱动的报告基因盒取代转座子携带的标记基因盒的可行性，这项工作将提供基本信息和技术，将广泛适用于当代植物基因组研究。 将要制造的结构和将要开发的技术都将在其他工厂中有用。

Deletional mutagenesis has not been used as extensively as insertional mutagenesis in Arabidopsis because large deletions are often not transmitted through gametes or are lethal early in development. The investigator will carry out a systematic study of small deletions by controlling their chromosomal location, developmental timing, and parent of origin. She will make deletions of known sizes using the site-specific bacteriophage P1-encoded Cre recombinase to delete chromosomal segments between a loxP-containing T-DNA donor site and a nearby loxP-containing transposon. Using an already tested transposon "launching pad" containing loxP recognition sites for the Cre recombinase, she will identify 3-4 transposons reinserted at different distances ranging from a few kilobases to a few megabases from each of several transposon donor sites to carry out these studies. She will use Cre recombinase genes expressed from a constitutive plant promoter, as well as from a chemically inducible promoter. She will detect deletions (and inversions) by both PCR-based and genetic methods. She will determine the genetic transmissibility of deletions and analyze the development of plants in which deletions are induced at different times after germination. This work will both increase the understanding of the detrimental effects of deletions and deletional heterozygosity and provide methods to control the size, chromosomal location, and timing of deletions.The second objective is to lay the groundwork for development of a transposon-based method to target genes to their original chromosomal locations. This technique, which the investigator calls gene "homing" to distinguish it from homology-based gene targeting, will use site-specific recombination to target a promoter-reporter cassette to a transposon-disrupted gene. If successful, this work will make it possible for the first time to study precise alterations in a plant gene's regulatory sequences in the gene's original chromatin context. The gene homing method will use transposons to target genes to their original chromatin environment by replacing a loxP-bracketed marker gene on the transposon with a promoter-reporter gene cassette. She will use existing plants with loxP transposons and transposon launching pads to determine whether an efficient "cassette replacement" technique developed in animal cells can also be used to integrate DNA segments efficiently between pairs of loxP sites in Arabidopsis. This work will establish the feasibility of replacing a transposon-borne marker gene cassette with a cassette carrying a reporter gene driven by the promoter of the disrupted gene.This work will provide basic information and techniques that will be widely applicable in contemporary plant genomic research. Both the constructs that will be made and the techniques that will be developed will be useful in other plants.