Understanding the mechanism of homologous recombination mediated gene targeting in Physcomitrella patens

陆地植物是静态生物，它们的生存取决于它们承受各种环境压力的能力。人类活动对环境的影响对这些压力的严重程度产生了不利影响，从而限制了作物生产力。臭氧消耗导致电离辐射暴露增加，全球气温升高和可用水量减少导致干旱压力增加。这些压力导致“活性氧”的产生：在细胞中积累并与细胞成分发生反应以使其失活或破坏其功能的高活性化学物质。一个非常容易受到这种损害的关键过程是遗传物质的维护。DNA是一种编码遗传信息的分子，对活性氧的破坏非常敏感。最严重的损伤形式是DNA双螺旋主干的断裂。如果不进行修复，这些断裂会导致遗传物质不可逆转的灾难性损失和随后的细胞死亡。为了解决这个问题，所有生物都进化出了高效的机制来修复这种DNA双链断裂（DNA- dsbs）。DNA-DSB修复主要有两种机制。一种是“快速而肮脏”的程序，称为“非同源末端连接”（NHEJ），它捕获DNA分子的断裂末端并将它们重新连接起来。然而，这个过程是不准确的，并且在修复位点包含DNA序列错误。第二种机制捕获断裂的末端，并通过复制同源序列精确地修复它们。这一过程（“同源重组”- HR）是没有错误的，当精子或卵细胞通过减数分裂产生时，也用于母系和父系染色体之间的遗传物质交换。这一过程负责种群内遗传多样性的产生，并在植物育种中被利用来将理想的性状引入新的作物品种。基因工程师也利用了DNA修复机制。当外源基因（转基因）被宿主细胞的DNA修复机制捕获后，被整合到宿主的基因组中，并通过NHEJ途径随机整合，或通过hr介导的途径在特定位点整合。hr介导的转基因整合发生在以下情况下：(i)转基因携带与基因组中靶位点相同的序列；（ii）宿主细胞表现出hr介导的修复优于nhej介导的修复。很少有生物优先使用HR途径进行DNA修复和转基因整合。在这样的情况下，有可能通过“基因靶向”（Gene Targeting，简称GT）进行精确的基因工程。在没有非特异性基因组改变的情况下，通过这种方法可以可靠地改变宿主基因的单个碱基对。由于其高度的精度，GT的部署将是作物改良战略的一个有吸引力的选择。目前，唯一能发生高效hr介导的GT的植物是藓类小立藓（Physcomitrella patens），这是第一个完成基因组测序的非开花陆地植物，也是研究植物基因功能进化的模型。由于苔藓中的GT是常规和有效的，它提供了一个理想的模型，以确定这一重要DNA修复途径的分子机制。本研究将鉴定和描述指导高效hr介导的GT的关键植物基因。这将为以下方面提供必要的基本理解：(1)基于知识的作物物种GT率的提高，这是“清洁”基因工程的先决条件；（ii）鉴定可以增强对dna破坏环境胁迫的抗性的基因；（iii）鉴定可以修改的HR机制的组成部分，以提高减数分裂衍生的遗传变异率，从而加速植物育种。

Land plants are static organisms, and their survival depends on their ability to withstand a variety of environmental stresses. The environmental impact of human activities is adversely affecting the severity of these stresses, and consequently limiting crop productivity. Ozone depletion leads to increased exposure to ionising radiation, and globally increasing temperatures and decreased water availability causing increased levels of drought stress. These stresses cause the generation of 'active oxygen species': highly reactive chemical agents that accumulate in cells and react with cellular components to inactivate or disrupt their functions. One key process that is highly susceptible to such damage is the maintenance of the genetic material. DNA, the molecule that encodes genetic information, is highly sensitive to damage by active oxygen species. The most severe forms of damage are breaks in the backbone of the DNA double-helix. If not repaired, these breaks result in irreversible and catastrophic loss of genetic material and subsequent cell death. To counter this, all organisms have evolved highly efficient mechanisms for the repair of such DNA double-strand breaks (DNA-DSBs). There are two principal mechanisms that are used for DNA-DSB repair. One is a 'quick and dirty' procedure called 'non-homologous end-joining' (NHEJ) that captures broken ends of DNA molecules and rejoins them. However, this process is inaccurate and incorporates DNA sequence errors at repair sites. The second mechanism captures broken ends and repairs them accurately by copying an homologous sequence. This process ('homologous recombination' - HR) is error-free, and is also used in the exchange of genetic material between maternal and paternal chromosomes when sperm or egg cells are produced by meiotic (reduction) division. This is process is responsible for the generation of genetic diversity within populations, and is exploited in plant breeding to introduce desirable traits into new crop varieties. DNA repair mechanisms are also exploited by genetic engineers. Delivery of a foreign gene (a transgene) into a cell results in its being integrated into the host's genome when it is captured by the host cell's DNA repair machinery and integrated either randomly, by the NHEJ pathway, or at a specific site by the HR-mediated pathway. HR-mediated transgene integration occurs if (i) the transgene carries sequences identical to a target site in the genome and (ii) if the host cell displays a preference for HR-mediated repair over NHEJ-mediated repair. Very few organisms preferentially use the HR pathway for DNA repair and transgene integration. In those that do, it is possible to undertake precision engineering of genes by 'Gene Targeting' (GT). As little as a single base-pair of a host gene can be reliably altered by this means, without non-specific alteration of the genome. Because of its high degree of precision, the deployment of GT would be an attractive option for crop improvement strategies. Currently, the only plant in which efficient HR-mediated GT occurs is a moss, Physcomitrella patens, the first non-flowering land plant to have its genome completely sequenced and a model for studies of the evolution of plant gene function. Because GT in moss is routine and efficient, it provides an ideal model in which to identify the molecular mechanisms underlying this important DNA repair pathway. This research will identify and characterize key plant genes that direct efficient HR-mediated GT. This will provide the fundamental understanding necessary for (i) knowledge-based enhancement of GT rates in crop species, a prerequisite for 'clean' genetic engineering; (ii) identification of genes that can enhance resistance to DNA-damaging environmental stresses and (iii) identification of components of the HR machinery that could be modified to generate enhanced rates of meiotically-derived genetic variation for accelerated plant breeding.