Transposon-mediated gene exploration in barley and other cereals

大麦和其他谷物中转座子介导的基因探索

• 批准号: RGPIN-2015-06652
• 负责人: Singh, Jaswinder
• 金额: $ 2.04万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=665286
• 关键词: Transposon; mediated; gene; exploration; barley

Characterization of mutant alleles provides valuable insights into the genetics of normal physiological functions. Mutagenesis via T-DNA insertion, RNA interference and transposon tagging enable researchers to more readily study specific genes and their functions. Transposon -mediated insertional mutagenesis has advantages over other approaches for determining gene function especially where genetic transformation is not routine. Important genes involved in development were identified using several gene-tagging strategies involving Ac/Ds-mediated insertional mutagenesis. During the transposon movement, the transposon inserts into a new chromosomal location and may cause interruption in gene's normal function. Some epialleles undergo paramutation which is an interaction between alleles and leads to a heritable expression change of one allele. Transposons have also served as building blocks for epigenetic phenomena, both at the level of single genes and across larger chromosomal regions. Studies are being conducted for the understanding these silencing mechanisms.*******Using transposon-based strategy, we have created over 400 unique transposon insertion lines (TNPs) in barley. Barley is a model genome system for Triticeae. Because barley is diploid, observation of the segregation of phenotypic variation in tagged mutants in successive generations is simpler than in its close polyploid relatives, wheat and oats. The system was tested in barley for several generations by reactivating Ds transposons and development of primary, secondary, tertaiary and quaternary transposon insertion lines. We also established that in barley, Ds transposon have tendency to move into genic regions in high frequency. The system is now ready for its use in functional genomic studies. The long-term objective of my research program is to advance molecular knowledge related to the mechanisms of seed dormancy and stress tolerance in cereals. From our TNP repository, we have identified transposon mutants of the DNA silencing pathway with reduced germination. There is emerging evidence that epigenetic may contribute to grain development and seed dormancy. Our preliminary characterization of transposon mutants indicate that DNA silencing pathway may also be involved in regulation of seed dormancy. Our recent data indicate that a key gene of RdDM pathway, AGO4 acts as negative regulator of dormancy. However, it is not known whether specific dormancy related genes are subjected to silencing through RdDM in small grain cereals. This may be due to DNA methylation triggered by small RNAs which needs further investigation. I propose to determine, the unknown role of (DCL3) in grain dormancy in barley through transposon mutagenesis. Data from these experiments will advance fundamental knowledge on the molecular mechanisms involved in the regulation of seed dormancy and germination.******

突变等位基因的表征为正常生理功能的遗传学提供了有价值的见解。通过T-DNA插入、RNA干扰和转座子标签的突变使研究人员能够更容易地研究特定基因及其功能。转座子介导的插入突变在确定基因功能方面比其他方法具有优势，特别是在遗传转化不是常规的情况下。利用几种涉及Ac/Ds介导的插入诱变的基因标记策略鉴定了参与发育的重要基因。在转座子运动过程中，转座子插入到一个新的染色体位置，并可能导致基因的正常功能中断。一些表观等位基因发生副突变，这是等位基因之间的相互作用，并导致一个等位基因的遗传表达变化。转座子还充当表观遗传现象的构建块，无论是在单个基因水平还是在更大的染色体区域。目前正在进行研究，以了解这些沉默机制。**利用基于转座子的策略，我们已经在大麦中创建了400多个独特的转座子插入系（TNP）。大麦是小麦族的模式基因组系统。由于大麦是二倍体，观察标记突变体在连续世代中的表型变异分离比其近多倍体亲属小麦和燕麦更简单。该系统在大麦中进行了几代测试，通过重新激活Ds转座子和初级，二级，三级和四级转座子插入系的发展。我们还证实，在大麦中，Ds转座子有向基因区移动的趋势，频率很高。该系统现已准备好用于功能基因组研究。我的研究计划的长期目标是推进与谷物种子休眠和胁迫耐受性机制相关的分子知识。从我们的TNP库，我们已经确定了转座子突变体的DNA沉默途径与减少发芽。越来越多的证据表明，表观遗传可能有助于谷物发育和种子休眠。我们对转座子突变体的初步鉴定表明，DNA沉默途径也可能参与调节种子休眠。 我们最近的研究表明，RdDM途径的关键基因AGO 4是休眠的负调控基因。然而，目前还不知道特定的休眠相关基因是否受到沉默通过RdDM在小粒谷物。这可能是由于小RNA引发的DNA甲基化，需要进一步研究。我建议确定，未知的作用（DCL 3）在大麦籽粒休眠通过转座子诱变。这些实验的数据将推进对种子休眠和萌发调控的分子机制的基础知识。