Controlled Deletional Mutagenesis and Gene Homing in Arabidopsis

由于大量的缺失通常不通过配子传递，或者在发育早期是致命的，所以在拟南芥中，删除突变并没有像插入突变那样被广泛使用。研究人员将通过控制染色体位置、发育时间和起源父母对微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重组酶删除已知大小的片段，以删除含有loxP的T-DNA供体部位和附近含有loxP的转座子之间的染色体片段。使用已经测试过的包含Cre重组酶loxP识别位点的转座子“发射台”，她将从几个转座子供体位置中的每个位置识别重新插入的3-4个转座子，距离从几千碱基到几兆碱基不等，以进行这些研究。她将使用从构成植物启动子和化学诱导启动子表达的Cre重组酶基因。她将通过基于聚合酶链式反应和遗传的方法来检测缺失(和反转)。她将确定缺失的遗传遗传性，并分析在萌发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了解，并提供控制缺失大小、染色体位置和缺失时机的方法。第二个目标是为开发基于转座子的方法将基因定位到其原始染色体位置奠定基础。这项技术被研究人员称为基因“归位”，以区别于基于同源的基因打靶，它将使用定点重组将启动子-报告盒靶向转座子破坏的基因。如果成功，这项工作将首次使研究植物基因在基因原始染色质背景下的调控序列的精确变化成为可能。基因归位方法将使用转座子将转座子上的loxP标记基因替换为启动子-报告基因盒，从而将基因靶向其原始染色质环境。她将利用现有的带有loxP转座子和转座子发射台的植物来确定在动物细胞中开发的一种有效的“盒式替换”技术是否也可以用于在拟南芥的loxP位点对之间有效地整合DNA片段。这项工作将确定用携带报告基因的转座子携带的标记基因盒取代转座子携带的标记基因盒的可行性，这项工作将为当代植物基因组研究提供广泛应用的基本信息和技术。将要制造的结构和将要开发的技术都将在其他工厂中使用。

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.