Functional analysis of the gene albostrians in barley and its impact on variegation and chloroplast biogenesis (Acronym: ALBOSTRIANS)

大麦基因 albostrians 的功能分析及其对杂色和叶绿体生物发生的影响（缩写：ALBOSTRANS）

• 批准号: 317066570
• 负责人: Dr. Jochen Kumlehn
• 金额: --
• 依托单位: Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/317066570?language=en
• 关键词: Functional; analysis; gene; albostrians; barley

The albostrians mutant shows a characteristically variegated leaf phenotype and it is one of the best characterized mutants of barley (Hordeum vulgare L.). The albostrians specific variegation phenotype is not fully transmissive, hence homozygous mutant plants may exhibit a wild-type phenotype. Progeny of such a plant will segregate for wildtype green (10%), albino (10%) and variegated (80%) plants. Studies on albostrians barley led to the discovery of plastid-to-nucleus (retrograde) signaling and of the nuclear-gene encoded plastid RNA polymerase NEP. Studies on albostrians clearly demonstrated that functional plastids/chloroplasts are important for plant-pathogen interactions and that the genetic apparatus of mitochondria responds to the developmental / metabolic state of chloroplasts. In a previous, core funded, project phase we isolated the underlying single nuclear gene of barley. We could confirm the gene function by genetic linkage and independent mutant allele analysis. Allocation of the putative protein to chloroplasts could be shown transiently by co-localization experiments of a GFP tagged albostrians gene and an YFP labeled chloroplast marker. Since the gene albostrians is potentially an important factor involved in non-reversible chloroplast maturation during early embryogenesis we aim to perform functional analysis of the newly identified gene in order to reach a better understanding of the gene function, which is of particular interest for basic and applied plant sciences. We want to obtain evidence for the hypothesis that this gene is not a plastome mutator as originally suggested and does not affect the chloroplast DNA but is involved directly or indirectly in the biogenesis of plastid ribosomes. We want to find out further whether a threshold of albostrians function is involved in the variegated leaf phenotype.We will perform:Functional validation of the identified albostrians gene by transgene analysis: Overexpression of the WT gene in WT and MT genetic background; Genome editing of WT albostrians by RNAguided/Cas9 to test the threshold hypothesis of residual gene function in differently truncated proteins; Heterologous complementation of Arabidopsis cia2/cia2-like mutants with the barley albostrians gene.Determination of ultrastructural differences between wild-type (As/As, As/as) and mutant albostrians plants; intraorganellar localization of the ALBOSTRIANS protein to determine at what point of development the dysfunction of ALBOSTRIANS leads to the lack of plastid ribosomes and to variegation?Unravelling the interactory network that involves the albostrians gene product by: CHipSEQ supported by HA-tagged albostrians protein; Quantitative RNAseq; Yeast-two-hybrid analysis; TILLING analysis of the barley paralog of the albostrians gene.

albostrians突变体表现出特征性的杂色叶表型，是大麦（Hordeum vulgare L.）的最佳特征突变体之一。Albostrians特异性杂色表型不是完全传递的，因此纯合突变体植物可以表现出野生型表型。这种植物的后代将分离出野生型绿色（10%）、白化病（10%）和杂色（80%）植物。对Albostrians大麦的研究发现了质体到核（逆行）信号传导和核基因编码的质体RNA聚合酶NEP。对albostrians的研究清楚地表明，功能性质体/叶绿体对植物-病原体相互作用是重要的，并且线粒体的遗传装置响应叶绿体的发育/代谢状态。在先前的核心资助项目阶段，我们分离了大麦的潜在单核基因。通过遗传连锁分析和独立突变等位基因分析，可以确定该基因的功能。通过GFP标记的albostrians基因和YFP标记的叶绿体标记物的共定位实验，可以瞬时显示推定蛋白质在叶绿体中的分配。由于基因albostrians是潜在的一个重要因素，在不可逆的叶绿体成熟在早期胚胎发生过程中，我们的目标是进行功能分析的新发现的基因，以达到更好地了解基因的功能，这是特别感兴趣的基础和应用植物科学。我们希望获得的假设，该基因是不是一个plastome mutator最初建议，不影响叶绿体DNA，但直接或间接参与质体核糖体的生物合成的证据。我们想进一步了解白化病功能阈值是否与杂色叶表型有关。我们将进行：通过转基因分析对已鉴定的白化病基因进行功能验证：WT基因在WT和MT遗传背景中的过表达;通过RNA引导/Cas9对WT白化病基因组进行编辑，以检验不同截短蛋白中残留基因功能的阈值假设;拟南芥cia 2/cia 2-like突变体与大麦albostrians基因的异源互补：野生型与野生型之间超微结构差异的测定（As/As，As/as）和突变体albostrians植物;蛋白质的细胞器内定位，以确定在发育的什么时候蛋白质的功能障碍导致质体核糖体的缺乏和杂色？通过HA标记的albostrians蛋白支持的CHipSEQ;定量RNAseq;酵母双杂交分析; albostrians基因的大麦partial的TILLING分析来解开涉及albostrians基因产物的相互作用网络。