NADPH依赖的烯醛氧化还原酶提高玉米耐低氮能力的机制分析

Lipid-derived reactive carbonyl species (RCS) increased as a result of the enhancement of lipid peroxidation when plants were subjected to low nitrogen and other stress conditions. Lipid-derived RCS altered the structure and function of cellular macromolecules, such as protein and DNA, subsequently leading to cell dysfunction. Our previous research showed that overexpression of the NADPH-dependent 2-alkenal reductase gene (445 gene) significantly increased the tolerance of maize to low nitrogen stress. In the present proposal, transgenic maize overexpression 445 gene and mutation plants constructed by CRISPR-Cas9 are used to analyze the effects of 445 gene on the contents of lipid-derived RCS and the degree of lipid peroxidation under low nitrogen stress at the cellular level. The activities of key enzymes in nitrogen and carbon metabolism are analyzed to evaluate the biochemical role of the 445 gene. RNA-seq study is used to resolve the effects of the 445 gene on nitrogen metabolism and to construct a preliminary regulatory network around the 445 gene illustrating its effect on the tolerance of maize to low nitrogen stress. The current work strongly suggests the mechanism by which overexpression of NADPH-dependent 2-alkenal reductase of enhances the tolerance of maize to low nitrogen stress. Moverover, the relationship of nitrogen use efficiency to lipid-derived RCS contents and 2-alkenal reductase activities will be revealed.

植物在低氮等逆境胁迫下细胞脂质过氧化反应增强，脂源性不饱和羰基化合物（RCS）含量增加，影响细胞内蛋白质、DNA等生物大分子的结构和功能，进而导致细胞功能紊乱。课题组前期研究发现在玉米中超量表达NADPH依赖的烯醛氧化还原酶基因（445基因）可以显著提高玉米的耐低氮能力。本研究利用445基因超量表达和CRISPR-Cas9突变体玉米植株从细胞水平分析低氮胁迫下445基因对细胞脂源性RCS含量和脂质过氧化程度的影响；从生化角度分析445基因对氮、碳代谢关键酶活性的影响；利用RNA-Seq技术从分子水平解析445基因对氮代谢的影响，初步构建445基因影响玉米耐低氮能力的调控网络。通过上述工作阐明NADPH依赖的烯醛氧化还原酶调节玉米耐低氮能力的机制，揭示植物细胞RCS含量以及烯醛氧化还原酶活性提高与氮高效的之间关系。

植物在低氮（LN）等逆境胁迫下细胞脂质过氧化反应增强，脂源性不饱和羰基化合物（RCS）含量增加，影响细胞内蛋白质、DNA等生物大分子的结构和功能，进而导致细胞功能紊乱。植物体内含有一系列可清除脂源性RCS的还原酶，降低不饱和羰基化合物（RCS）对细胞的毒害。然而，这些酶在营养胁迫下减轻氧化胁迫和提高营养利用效率（NUE）的作用仍然未知。我们在玉米中过表达玉米内源NADPH依赖性2-烯醛还原酶（ZmAER），发现显著提高了转基因植株（OX-AER）对低氮胁迫的耐受性。在低氮（LN）条件下，转基因植株（OX-AER）的生物量、氮积累、NUE和叶片光合作用均显着高于野生型（WT）植株。转基因植株的叶和根中丙二醛和H2O2水平明显低于野生型植物。抗氧化酶相关基因ZmCAT3、ZmPOD5和ZmPOD13的表达在转基因系中显著高于WT。在低氮（LN）胁迫下，与野生型（WT）叶片相比，转基因植株（OX-AER）叶片中的硝酸还原酶活性显著增加。此外，在低氮（LN）胁迫下，与野生型（WT）植株相比，转基因植株（OX-AER）中ZmNRT1.1和ZmNRT2.5的表达上调。总体而言，上调的ZmAER表达可以通过减轻氧化胁迫和改善NUE来增强玉米对低氮胁迫的耐受性。研究结果可能为作物氮高效的理论研究提供新的思路，为氮高效玉米的培养提供新种质。