Dunaliella salina can produce glycerol under salt stress, and this production can quickly adapt to changes in external salt concentration. Notably, glycerol is an ideal energy source. In recent years, it has been reported that the mitogen-activated protein kinase cascade pathway plays an important role in regulating salt stress, and in Dunaliella tertiolecta DtMAPK can regulate glycerol synthesis under salt stress. Therefore, it is highly important to study the relationship between the MAPK cascade pathway and salt stress in D. salina and modify it to increase the production of glycerol. In our study, we identified and analysed the alternative splicing of DsMEK1 (DsMEK1-X1, DsMEK1-X2) from the unicellular green alga D. salina. DsMEK1-X1 and DsMEK1-X2 were both localized in the cytoplasm. qRT-PCR assays showed that DsMEK1-X2 was induced by salt stress. Overexpression of DsMEK1-X2 revealed a higher increase rate of glycerol production compared to the control and DsMEK1-X1-oe under salt stress. Under salt stress, the expression of DsGPDH2/3/5/6 increased in DsMEK1-X2-oe strains compared to the control. This finding indicated that DsMEK1-X2 was involved in the regulation of DsGPDH expression and glycerol overexpression under salt stress. Overexpression of DsMEK1-X1 increased the proline content and reduced the MDA content under salt stress, and DsMEK1-X1 was able to regulate oxidative stress; thus, we hypothesized that DsMEK1-X1 could reduce oxidative damage under salt stress. Yeast two-hybrid analysis showed that DsMEK1-X2 could interact with DsMAPKKK1/2/3/9/10/17 and DsMAPK1; however, DsMEK1-X1 interacted with neither upstream MAPKKK nor downstream MAPK. DsMEK1-X2-oe transgenic lines increased the expression of DsMAPKKK1/3/10/17 and DsMAPK1, and DsMEK1-X2-RNAi lines decreased the expression of DsMAPKKK2/10/17. DsMEK1-X1-oe transgenic lines did not exhibit increased gene expression, except for DsMAPKKK9. Our findings demonstrate that DsMEK1-X1 and DsMEK1-X2 can respond to salt stress by two different pathways. The DsMEK1-X1 response to salt stress reduces oxidative damage; however, the DsMAPKKK1/2/3/9/10/17-DsMEK1-X2-DsMAPK1 cascade is involved in the regulation of DsGPDH expression and thus glycerol synthesis under salt stress.

中文翻译：

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DsMEK1 丝裂原活化蛋白激酶激酶 (MAPKK) 的两个剪接变体以不同方式参与了盐生杜氏藻的盐胁迫调节。

盐胁迫下杜氏盐藻能产生甘油，这种产生能迅速适应外界盐浓度的变化。值得注意的是，甘油是一种理想的能源。近年来，有报道称丝裂原活化蛋白激酶级联通路在调节盐胁迫中起重要作用，而Dunaliella tertiolecta DtMAPK可以调节盐胁迫下的甘油合成。因此，研究 MAPK 级联途径与盐胁迫之间的关系并对其进行修改以增加甘油的产量非常重要。在我们的研究中，我们从单细胞绿藻 D. salina 中鉴定并分析了 DsMEK1（DsMEK1-X1，DsMEK1-X2）的可变剪接。DsMEK1-X1 和 DsMEK1-X2 均位于细胞质中。qRT-PCR 分析表明 DsMEK1-X2 是由盐胁迫诱导的。在盐胁迫下，与对照和 DsMEK1-X1-oe 相比，DsMEK1-X2 的过表达显示甘油产量的增加率更高。在盐胁迫下，与对照相比，DsMEK1-X2-oe 菌株中 DsGPDH2/3/5/6 的表达增加。这一发现表明 DsMEK1-X2 参与了盐胁迫下 DsGPDH 表达和甘油过表达的调节。DsMEK1-X1过表达盐胁迫下脯氨酸含量增加，MDA含量降低，DsMEK1-X1能够调节氧化应激；因此，我们假设 DsMEK1-X1 可以减少盐胁迫下的氧化损伤。酵母双杂交分析表明，DsMEK1-X2可以与DsMAPKKK1/2/3/9/10/17和DsMAPK1相互作用；然而，DsMEK1-X1 既不与上游 MAPKKK 也不与下游 MAPK 相互作用。DsMEK1-X2-oe转基因株系增加了DsMAPKKK1/3/10/17和DsMAPK1的表达，而DsMEK1-X2-RNAi株系降低了DsMAPKKK2/10/17的表达。DsMEK1-X1-oe 转基因系没有表现出增加的基因表达，除了 DsMAPKKK9。我们的研究结果表明，DsMEK1-X1 和 DsMEK1-X2 可以通过两种不同的途径对盐胁迫作出反应。DsMEK1-X1 对盐胁迫的反应减少了氧化损伤；然而，DsMAPKKK1/2/3/9/10/17-DsMEK1-X2-DsMAPK1 级联参与调节 DsGPDH 表达，从而在盐胁迫下参与甘油合成。DsMEK1-X1-oe 转基因系没有表现出增加的基因表达，除了 DsMAPKKK9。我们的研究结果表明，DsMEK1-X1 和 DsMEK1-X2 可以通过两种不同的途径对盐胁迫作出反应。DsMEK1-X1 对盐胁迫的反应减少了氧化损伤；然而，DsMAPKKK1/2/3/9/10/17-DsMEK1-X2-DsMAPK1 级联参与调节 DsGPDH 表达，从而在盐胁迫下参与甘油合成。DsMEK1-X1-oe 转基因系没有表现出增加的基因表达，除了 DsMAPKKK9。我们的研究结果表明，DsMEK1-X1 和 DsMEK1-X2 可以通过两种不同的途径对盐胁迫作出反应。DsMEK1-X1 对盐胁迫的反应减少了氧化损伤；然而，DsMAPKKK1/2/3/9/10/17-DsMEK1-X2-DsMAPK1 级联参与调节 DsGPDH 表达，从而在盐胁迫下参与甘油合成。