Transposon-mediated gene exploration in barley and other cereals

突变等位基因的特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干扰和转座子标记的诱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比其他确定基因功能的方法具有优势，特别是在遗传转化不常规的情况下。通过几种基因标记策略，包括Ac/ ds介导的插入突变，确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，可能导致基因正常功能的中断。一些外显等位基因发生参数化，这是等位基因之间的相互作用，导致一个等位基因的遗传表达变化。转座子也作为表观遗传现象的组成部分，无论是在单基因水平上还是在更大的染色体区域上。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，我们在大麦中创建了400多个独特的转座子插入系（TNPs）。大麦是小麦科的典型基因组系统。由于大麦是二倍体，在连续代中观察标记突变体的表型变异比在其多倍体近亲小麦和燕麦中更简单。该系统在大麦中进行了几代的试验，通过重新激活Ds转座子，并培养了一级、二级、三级和四级转座子插入系。我们还发现，在大麦中，Ds转座子有向基因区高频率移动的趋势。该系统现在已经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抗逆性机制相关的分子知识。从我们的TNP库中，我们已经确定了DNA沉默途径的转座子突变体，其萌发率降低。越来越多的证据表明，表观遗传可能与谷物发育和种子休眠有关。我们对转座子突变体的初步表征表明，DNA沉默途径也可能参与了种子休眠的调控。我们最近的数据表明，RdDM通路的关键基因AGO4在休眠中起负调控作用。然而，在小粒谷物中，特定的休眠相关基因是否通过RdDM被沉默尚不清楚。这可能是由于小rna引发的DNA甲基化，这需要进一步研究。我建议通过转座子诱变来确定（DCL3）在大麦籽粒休眠中的未知作用。这些实验的数据将促进对参与调节种子休眠和发芽的分子机制的基本认识。******

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.******