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

了解小立碗藓同源重组介导的基因靶向机制

基本信息

  • 批准号:
    BB/I006710/1
  • 负责人:
  • 金额:
    $ 58.82万
  • 依托单位:
  • 依托单位国家:
    英国
  • 项目类别:
    Research Grant
  • 财政年份:
    2011
  • 资助国家:
    英国
  • 起止时间:
    2011 至 无数据
  • 项目状态:
    已结题

项目摘要

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.
陆地植物是静止的生物,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性产生了不利影响,从而限制了作物的生产力。臭氧消耗导致电离辐射暴露增加,全球气温上升,水供应减少,导致干旱压力增加。这些压力导致“活性氧物质”的产生:高度反应性的化学物质在细胞中积累并与细胞成分反应以破坏或破坏其功能。一个非常容易受到这种损害的关键过程是遗传物质的维持。DNA是编码遗传信息的分子,对活性氧的破坏高度敏感。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损失以及随后的细胞死亡。为了应对这一点,所有生物都进化出了高效的DNA双链断裂修复机制(DNA-DSB)。有两种主要机制用于DNA-DSB修复。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其重新连接起来。然而,这一过程是不准确的,并在修复位点掺入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来精确地修复它们。这个过程(“同源重组”- HR)是无错误的,并且也用于当精子或卵细胞通过减数分裂(减数分裂)产生时,母本和父本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,以将所需的性状引入新的作物品种。DNA修复机制也被遗传工程师利用。当外源基因(转基因)被宿主细胞的DNA修复机制捕获并通过NHEJ途径随机整合或通过HR介导的途径在特定位点整合时,将外源基因(转基因)递送到细胞中导致其整合到宿主基因组中。如果(i)转基因携带与基因组中的靶位点相同的序列,并且(ii)如果宿主细胞显示出HR介导的修复优于NHEJ介导的修复,则发生HR介导的转基因整合。很少有生物优先使用HR途径进行DNA修复和转基因整合。在那些这样做的人中,有可能通过“基因靶向”(GT)进行基因的精确工程。通过这种方法,可以可靠地改变宿主基因的单个碱基对,而不会对基因组造成非特异性改变。由于其高度的精确性,GT的部署将是作物改良战略的一个有吸引力的选择。目前,唯一的植物,其中有效的HR介导的GT发生是苔藓,小立碗藓,第一个非开花的陆地植物有其基因组完全测序和植物基因功能的进化研究的模型。由于GT在苔藓中是常规和有效的,它提供了一个理想的模型,以确定这一重要的DNA修复途径的分子机制。这项研究将确定和表征指导高效HR介导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)基于知识的作物GT率提高,这是“清洁”基因工程的先决条件;(ii)鉴定可以增强对DNA破坏性环境胁迫的抗性的基因,以及(iii)鉴定可以被修饰以产生用于加速植物育种的减数分裂衍生的遗传变异的增强速率的HR机制的组分。

项目成果

期刊论文数量(7)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
An ancestral stomatal patterning module revealed in the non-vascular land plant Physcomitrella patens.
  • DOI:
    10.1242/dev.135038
  • 发表时间:
    2016-09-15
  • 期刊:
  • 影响因子:
    0
  • 作者:
    Caine RS;Chater CC;Kamisugi Y;Cuming AC;Beerling DJ;Gray JE;Fleming AJ
  • 通讯作者:
    Fleming AJ
Origin and function of stomata in the moss Physcomitrella patens.
  • DOI:
    10.1038/nplants.2016.179
  • 发表时间:
    2016-11-28
  • 期刊:
  • 影响因子:
    18
  • 作者:
  • 通讯作者:
MRE11 and RAD50, but not NBS1, are essential for gene targeting in the moss Physcomitrella patens.
  • DOI:
    10.1093/nar/gkr1272
  • 发表时间:
    2012-04
  • 期刊:
  • 影响因子:
    14.9
  • 作者:
    Kamisugi Y;Schaefer DG;Kozak J;Charlot F;Vrielynck N;Holá M;Angelis KJ;Cuming AC;Nogué F
  • 通讯作者:
    Nogué F
Towards designer organelles by subverting the peroxisomal import pathway.
  • DOI:
    10.1038/s41467-017-00487-7
  • 发表时间:
    2017-09-06
  • 期刊:
  • 影响因子:
    16.6
  • 作者:
    Cross LL;Paudyal R;Kamisugi Y;Berry A;Cuming AC;Baker A;Warriner SL
  • 通讯作者:
    Warriner SL
The Transcriptional Response to DNA-Double-Strand Breaks in Physcomitrella patens.
  • DOI:
    10.1371/journal.pone.0161204
  • 发表时间:
    2016
  • 期刊:
  • 影响因子:
    3.7
  • 作者:
    Kamisugi Y;Whitaker JW;Cuming AC
  • 通讯作者:
    Cuming AC
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Andrew Cuming其他文献

Andrew Cuming的其他文献

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{{ truncateString('Andrew Cuming', 18)}}的其他基金

Doctoral Training Grant
博士培训补助金
  • 批准号:
    BB/F01578X/1
  • 财政年份:
    2009
  • 资助金额:
    $ 58.82万
  • 项目类别:
    Training Grant
Integrating the genome sequence and genetic linkage map of Physcomitrella patens: a platform for map based gene cloning
整合小立碗藓的基因组序列和遗传连锁图谱:基于图谱的基因克隆平台
  • 批准号:
    BB/F001797/1
  • 财政年份:
    2007
  • 资助金额:
    $ 58.82万
  • 项目类别:
    Research Grant

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