Controlled Deletional Mutagenesis and Gene Homing in Arabidopsis

在拟南芥中，缺失诱变并不像插入诱变那样广泛应用，因为大的缺失通常不会通过配子传播或在发育早期是致命的。研究者将通过控制小缺失的染色体位置、发育时间和亲本来进行系统的研究。她将使用位点特异性噬菌体p1编码的Cre重组酶进行已知大小的删除，以删除含有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。利用一个已经测试过的包含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点中找出3-4个以不同距离重新插入的转座子，从几千个碱基到几百万个碱基不等，以开展这些研究。她将使用从组成植物启动子和化学诱导启动子中表达的Cre重组酶基因。她将通过基于聚合酶链反应和遗传方法检测缺失（和反转）。她将确定缺失的遗传传递性，并分析在萌发后不同时间诱导缺失的植物的发育情况。这项工作将增加对缺失和缺失杂合性的有害影响的理解，并提供控制缺失大小、染色体位置和时间的方法。第二个目标是为开发一种基于转座子的方法奠定基础，将基因靶向到其原始染色体位置。这项技术被研究者称为基因“归巢”，以区别于基于同源的基因靶向，它将使用位点特异性重组将启动子报告磁带定位到转座子破坏的基因上。如果成功，这项工作将使首次研究基因原始染色质背景下植物基因调控序列的精确变化成为可能。基因归巢方法将利用转座子将基因定位到其原始染色质环境，方法是用启动子报告基因盒替换转座子上的loxp括号标记基因。她将使用现有的具有loxP转座子和转座子发射平台的植物来确定在动物细胞中开发的有效的“盒式替换”技术是否也可以用于在拟南芥中loxP位点对之间有效地整合DNA片段。这项工作将确定将转座子携带的标记基因盒替换为携带由被破坏基因启动子驱动的报告基因盒的可行性。这项工作将为当代植物基因组研究提供广泛应用的基础信息和技术。将会制造的结构和将会开发的技术将会在其他植物中有用。

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.