College of Grassland Science and Technology, China Agricultural University, Beijing, 100193, China.
Plant Cell Rep. 2023 Apr;42(4):805-819. doi: 10.1007/s00299-023-02993-z. Epub 2023 Feb 9.
Sequestering microRNA396 by overexpression of MIM396 enhanced alfalfa resistance to Spodoptera litura larvae, which may be due to increased lignin content and enhanced low-molecular weight flavonoids and glucosinolates biosynthesis. Alfalfa (Medicago sativa), the most important leguminous forage crop, suffers from the outbreak of defoliator insects, especially Spodoptera litura, resulting in heavy losses in yield and forage quality. Here, we found that the expression of alfalfa microRNA396 (miR396) precursor genes and mature miR396 was significantly up-regulated in wounding treatment that simulates feeding injury by defoliator insects. To verify the function of miR396 in alfalfa resistance to insect, we generated MIM396 transgenic alfalfa plants with significantly down-regulated miR396 expression by Agrobacterium-mediated genetic transformation. The MIM396 transgenic alfalfa plants exhibited improved resistance to Spodoptera litura larvae with increased lignin content but decreased JA accumulation. Most of the miR396 putative target GRF genes were up-regulated in MIM396 transgenic lines, and responded to the wounding treatment. By RNA sequencing analysis, we found that the differentially expressed genes related to insect resistance between WT and MIM396 transgenic plants mainly clustered in biosynthesis pathways in lignin, flavonoids and glucosinolates. In addition to the phenotype of enhanced insect resistance, MIM396 transgenic plants also displayed reduced biomass yield and forage quality. Our results broaden the function of miR396 in alfalfa and provide genetic resources for studying alfalfa insect resistance.
通过过量表达 MIM396 来隔离 microRNA396 增强了紫花苜蓿对斜纹夜蛾幼虫的抗性，这可能是由于木质素含量增加和低分子量类黄酮和硫代葡萄糖苷生物合成增强所致。紫花苜蓿（Medicago sativa）是最重要的豆科饲料作物，遭受食叶昆虫，特别是斜纹夜蛾的爆发，导致产量和饲料质量严重损失。在这里，我们发现紫花苜蓿 microRNA396（miR396）前体基因和成熟 miR396 的表达在模拟食叶昆虫取食损伤的创伤处理中显著上调。为了验证 miR396 在紫花苜蓿抗虫中的功能，我们通过农杆菌介导的遗传转化生成了 MIM396 转基因紫花苜蓿植物，其 miR396 表达显著下调。MIM396 转基因紫花苜蓿植物对斜纹夜蛾幼虫的抗性提高，木质素含量增加，但 JA 积累减少。MIM396 转基因系中大多数 miR396 假定靶标 GRF 基因上调，并对创伤处理有反应。通过 RNA 测序分析，我们发现 WT 和 MIM396 转基因植物之间与昆虫抗性相关的差异表达基因主要聚类在木质素、类黄酮和硫代葡萄糖苷生物合成途径中。除了增强的抗虫表型外，MIM396 转基因植物的生物量产量和饲料质量也降低。我们的研究结果拓宽了 miR396 在紫花苜蓿中的功能，并为研究紫花苜蓿的抗虫性提供了遗传资源。
