Transposon-mediated gene exploration in barley and other cereals

突变等位基因的特征为研究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干扰和转座子标签进行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更有优势，特别是在基因转化不是常规的情况下。通过几种涉及Ac/DS介导的插入突变的基因标记策略，确定了与发育有关的重要基因。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。一些表观等位基因经历了等位基因之间的相互作用，从而导致一个等位基因的可遗传表达变化。转座子也是表观遗传现象的基石，无论是在单基因水平上，还是在更大的染色体区域上。为了理解这些沉默机制，我们正在进行研究。*利用基于转座子的策略，我们已经在大麦中创造了400多个独特的转座子插入系(TNPs)。大麦是小麦族的一个模式基因组系统。由于大麦是二倍体，观察到标记突变体在连续世代中的表型变异分离比其近缘的多倍体亲缘小麦和燕麦更简单。该系统在大麦上进行了几个世代的试验，通过重新激活DS转座子和建立第一、第二、三元和第四代转座子插入系。我们还证实在大麦中，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促进与谷物种子休眠和逆境耐受性机制相关的分子知识。从我们的TNP库中，我们已经鉴定出发芽减少的DNA沉默途径的转座子突变体。越来越多的证据表明，表观遗传可能有助于籽粒发育和种子休眠。我们对转座子突变体的初步鉴定表明，DNA沉默途径可能也参与了种子休眠的调节。我们最近的数据表明，RdDM途径的关键基因AGO4是休眠的负调控基因。然而，目前还不清楚特定的休眠相关基因是否通过RdDM在小粒谷物中受到沉默。这可能是由于小RNA引发的DNA甲基化，需要进一步研究。我建议通过转座子突变来确定(DCL3)在大麦籽粒休眠中的未知作用。来自这些实验的数据将促进关于调节种子休眠和萌发的分子机制的基础知识。

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.******