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

陆地植物是静态的有机体，它们的生存依赖于它们抵抗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。臭氧消耗导致暴露于电离辐射的增加，全球气温上升和可用水减少导致干旱压力增加。这些压力会导致“活性氧物种”的产生：高活性的化学物质聚集在细胞中，并与细胞成分反应，使其失活或破坏其功能。对这种损害非常敏感的一个关键过程是遗传物质的维护。DNA是编码遗传信息的分子，对活性氧物种的损伤高度敏感。最严重的损伤形式是DNA双螺旋骨架的断裂。如果不修复，这些断裂会导致遗传物质的不可逆转和灾难性的损失，并随后导致细胞死亡。为了应对这一点，所有生物都进化出了修复这种DNA双链断裂(DNA-DSB)的高效机制。有两种主要的机制用于DNA-DSB修复。一种是一种被称为“非同源末端连接”(NHEJ)的“快速而肮脏”的过程，它捕获DNA分子的断端并重新连接它们。然而，这一过程是不准确的，并在修复部位包含了DNA序列错误。第二种机制捕获断裂末端，并通过复制同源序列准确地修复它们。这一过程(同源重组)是没有错误的，当精子或卵细胞通过减数分裂产生时，也用于母本和父本染色体之间的遗传物质交换。这一过程负责在种群内产生遗传多样性，并被用于植物育种，将理想的特征引入新的作物品种。DNA修复机制也被基因工程师所利用。将外源基因(转基因)送入细胞，当它被宿主细胞的DNA修复机制捕获并随机整合时，它会被整合到宿主的基因组中，或者通过NHEJ途径整合，或者通过HR介导的途径在特定位置整合。如果(I)转基因携带的序列与基因组中的目标位置相同，并且(Ii)如果宿主细胞表现出对HR介导的修复的偏好而不是NHEJ介导的修复，则发生HR介导的转基因整合。很少有生物优先利用HR途径进行DNA修复和转基因整合。在那些做到了这一点的国家，有可能通过“基因打靶”(GT)对基因进行精确工程。通过这种方法可以可靠地改变宿主基因的单个碱基对，而不会对基因组进行非特异性改变。由于GT的高度精确度，部署GT将是作物改良战略的一个有吸引力的选择。目前，唯一有效的HR介导GT的植物是苔藓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来确定这一重要的DNA修复途径的分子机制。这项研究将确定和表征指导有效的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：(I)以知识为基础提高作物物种的GT率，这是‘清洁’基因工程的先决条件；(Ii)识别可以增强对DNA破坏的环境胁迫的抵抗力的基因；以及(Iii)识别HR机制的组件，这些组件可以被修改以产生更高的减数分裂遗传变异率，以加速植物育种。

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.