图1 拟南芥种子萌发过程中AtR8 lncRNA的表达特性分析 (A) 拟南芥种子萌发过程中AtR8 lncRNA表达特性的Northern分析(以5.8S rRNA作为上样对照); (B) 拟南芥种子萌发过程中AtR8 lncRNA组织表达特性的整体原位杂交(箭头指示AtR8 lncRNA信号) (Bars=200 μm)。

Figure 1 Analysis of AtR8 lncRNA expression during seed germination in Arabidopsis thaliana (A) Northern blotting analysis of AtR8 lncRNA expression during seed germination (expression of 5.8S rRNA serves as loading controls); (B) In situ hybridization of AtR8 lncRNA expression during seed germination (arrow indicates AtR8 lncRNA signal) (Bars=200 μm).

图2 拟南芥种子萌发过程中AtR8 lncRNA表达响应NaCl逆境胁迫 (A) AtR8 lncRNA与UCC盐胁迫响应元件的序列比较分析(USE、TATA启动子序列为大写、加粗并加框, AtR8 lncRNA转录区域为大写并加粗, 保守的盐胁迫响应元件用星号标注)。(B) AtR8 lncRNA二级结构中盐胁迫响应元件存在位置的RNAlogo预测(盐胁迫响应元件为大写、加粗并用箭头指出)。(C) 拟南芥种子萌发过程中不同浓度NaCl处理下AtR8 lncRNA表达特性的Northern分析, 以5.8S rRNA作为上样对照。下方为Northern半定量分析, 2个独立的实验给出了相似的结果, 并显示了1个代表性的例子。值为平均值±标准误(t-检验, *P<0.05, **P<0.01)。(D) 拟南芥种子萌发过程中150 mmol·L-1 NaCl处理不同时间AtR8 lncRNA表达特性的Northern分析, 以5.8S rRNA作为上样对照。下方为Northern半定量分析, 2个独立的实验给出了相似的结果, 并显示了1个代表性的例子。值为平均值±标准误(t-检验, **P<0.01)。

Figure 2 AtR8 lncRNA expression during seed germination of Arabidopsis thaliana after NaCl treatment (A) Sequence comparison between AtR8 lncRNA and UCC salt stress-responsive element (the USE and TATA promoter sequences are capitalized, bloded and framed; the AtR8 lncRNA transcriptional region is capitalized and bolded; and the conserved salt stress-responsive element is marked with asterisk). (B) The location of the salt stress-responsive element in the secondary structure of AtR8 lncRNA predicted by RNAlogo (the salt stress-responsive motif is capitalized, bloded and indicated with an arrow). (C) Northern blotting analysis of AtR8 lncRNA expression in germinating seeds under different NaCl treatments, 5.8S rRNA was used as a loading control. The lower panel shows semi-quantitative analysis of the Northern blotting signals. Two independent experiments gave similar results, and a representative example is shown. Values are means ± SE (t-test, *P<0.05, **P<0.01). (D) Northern blotting analysis of AtR8 lncRNA in germinating seeds under different periods of 150 mmol·L-1 NaCl treatment, 5.8S rRNA was used as a loading control. The lower panel shows semi-quantitative analysis of the Northern blotting signals. Two independent experiments gave similar results, and a representative example is shown. Values are means ± SE (t-test, **P<0.01).

图3 盐胁迫下AtR8 lncRNA缺失抑制拟南芥种子萌发 (A) Northern分析鉴定AtR8 lncRNA缺失突变体atr8 (以5.8S rRNA作为上样对照); (B) 不同浓度NaCl处理下, 野生型和atr8种子生长状况及萌发率统计分析(数据为3次独立试验的平均值, 误差为标准误); (C) 不同浓度NaCl处理下, 野生型和atr8萌发7天种子的鲜重及干重(数值为3次独立试验的平均值, 误差为标准误, 星号表示atr8与野生型的显著性差异(t-检验, *P<0.05, **P<0.01))。WT: 野生型

Figure 3 Loss of AtR8 lncRNA inhibits Arabidopsis thaliana seed germination under salt stress (A) Northern blotting analysis of the AtR8 lncRNA loss-of-function mutant atr8 (5.8S rRNA was used as the loading control); (B) Statistical analysis of growth and germination rate of the wild-type and atr8 seeds under different concentrations of NaCl (data are average of three independent experiments, and bars indicate standard error); (C) Fresh and dry weight of the wild-type and atr8 seeds after 7 d of germination under NaCl treatment (the values are average of three independent experiments, and bars indicate standard error (the asterisk indicate significant differences between atr8 and wild type (t-test, * P < 0.05, ** P < 0.01)). WT: Wild type