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首页> 外文期刊>Journal of Experimental Botany >A transient assay system for the assessment of cell-autonomous gene function in dehydration-stressed barley.
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A transient assay system for the assessment of cell-autonomous gene function in dehydration-stressed barley.

机译：瞬态测定系统，用于评估脱水胁迫的大麦中细胞自主基因的功能。

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Drought is a serious, worldwide problem for crop production and also affects yields of barley and wheat, together with other stressors such as frost, viral diseases, or fungal pathogens. Although a number of candidate genes have been identified by transcriptome approaches in recent years, only very few have been tested in functional assays for a beneficial effect on drought tolerance. Here, a transient assay system in microprojectile-bombarded barley leaves is described that allows the functional testing of dehydration stress-related candidate genes by RNA interference (RNAi) or overexpression. Cellular stress or damage in dedydrated leaves is reported by a reduced accumulation of slowly maturing, native red-fluorescing protein DsRed that is known to be sensitive to denaturing conditions. After a dehydration-stress period of 4 d during which the relative fresh weight of leaves was kept at 60-66% of initial fresh weight, a reproducible reduction of normalized DsRed fluorescence was observed. In order to obtain proof of concept, a number of barley mRNAs homologous to drought response genes were selected and targeted by transient induced gene silencing (TIGS). TIGS of four tested genes resulted in a significantly stronger decrease of normalized DsRed fluorescence in dehydration-stressed leaves, whereas they had no effect in fully turgescent control leaves. These genes encode barley drought-responsive factor HvDRF1 (DREB2-like), dehydrin 6, late embryogenesis-abundant protein HVA1, and the vacuolar sodium/proton antiporter HvHNX1. The four targeted transcripts were also found to accumulate rapidly in dehydration-stressed barley leaf segments. The results suggest a value of the TIGS system for functional pre-screening of larger numbers of drought or dehydration stress-related candidate genes in barley.
机译：干旱是世界范围内严重的农作物生产问题，还影响大麦和小麦的产量，以及霜冻，病毒性疾病或真菌病原体等其他胁迫因素。尽管近年来已通过转录组方法鉴定出许多候选基因，但在功能测定中仅测试了极少数对耐旱性有益的基因。在此，描述了一种在微粒轰击的大麦叶中的瞬时测定系统，该系统允许通过RNA干扰（RNAi）或过表达来对脱水胁迫相关候选基因进行功能测试。据报道，缓慢成熟的天然红色荧光蛋白DsRed的积累减少，据称对脱去叶片的细胞产生应力或破坏，该蛋白对变性条件敏感。在脱水胁迫4天后，叶片的相对鲜重保持在初始鲜重的60-66％，观察到可归一化的归一化DsRed荧光减少。为了获得概念证明，选择了许多与干旱反应基因同源的大麦mRNA，并通过瞬时诱导基因沉默（TIGS）靶向。四个测试基因的TIGS导致脱水胁迫的叶片中归一化的DsRed荧光明显更强的降低，而它们在完全饱和的对照叶片中没有作用。这些基因编码大麦干旱反应因子HvDRF1（类似于DREB2），脱水蛋白6，晚期胚发生丰富蛋白HVA1和液泡钠/质子逆转运蛋白HvHNX1。还发现这四个靶向转录物在脱水胁迫的大麦叶片段中迅速积累。结果表明，TIGS系统对于大麦中大量干旱或与干旱胁迫相关的候选基因的功能预筛选具有重要意义。

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