Abstract: This study investigated the physiological and molecular mechanisms underlying the cold tolerance of Caucasian clover （Trifolium ambiguum M. Bieb.）， a perennial forage known for its strong hardiness. Seedlings grown at 20℃ served as the control （CK）， and low-temperature stress （4℃） was applied for 2， 6， and 12 h. Physiological indicators were measured at each time point， and transcriptome sequencing was conducted on the control and 12 h-stressed plants. The results indicated that under 4℃， the plants enhanced their cold tolerance by significantly increasing the activities of antioxidant enzymes （peroxidase， catalase， and superoxide dismutase） and the levels of osmoregulatory substances （soluble protein， soluble sugar， and free proline）. This coordinated response effectively scavenged reactive oxygen species and mitigated membrane lipid peroxidation， as evidenced by reduced malondialdehyde accumulation. Transcriptome analysis identified 15 428 differentially expressed genes （DEGs） after 12 h of cold stress， with 8909 up-regulated and 6519 down-regulated. Key pathways were significantly activated， including the flavonoid biosynthesis pathway， where the up-regulation of pivotal upstream genes （CHS and CHI） facilitated the synthesis of antioxidant compounds such as luteolin， apigenin， and isoflavones. Concurrently， the MAPK signaling pathway was induced， highlighted by the increased expression of genes like MEKK1， suggesting that cold signals are transduced via a phosphorylation cascade. These findings provide crucial insights into the molecular basis of cold adaptation in Caucasian clover.

Key words: Trifolium ambiguum, Low temperature stress, Physiological parameters, DGEs

摘要： 本研究以耐寒性强的多年生牧草高加索三叶草（Trifolium ambiguum M. Bieb.）为材料，以20℃正常生长的幼苗为对照（CK），设置4℃低温胁迫2 h，6 h 和 12 h共3个处理，测定各时期高加索三叶草的生理指标，并对CK与胁迫12 h的植株进行转录组测序，解析其幼苗期在低温胁迫下的生理反应与适应机制。结果表明，4℃处理下，植株通过提高抗氧化酶（过氧化物酶、过氧化氢酶、超氧化物歧化酶）活性及渗透调节物质（可溶性蛋白、可溶性糖、游离脯氨酸）含量，有效清除活性氧，减轻了膜脂过氧化损伤，从而增强其抗寒能力。转录组分析显示，4℃处理12 h后共鉴定到15 428个差异表达基因，其中8909个基因上调，6519个基因下调。其中类黄酮生物合成通路与MAPK信号通路被显著激活：类黄酮通路上游关键基因查尔酮合成酶和查尔酮异构酶显著上调，促进了抗氧化物质（木犀草素、芹菜素、异黄酮）的合成；MAPK通路中MEKK1等基因的表达升高，表明低温信号通过磷酸化级联反应进行转导。其研究结果为解析高加索三叶草耐寒性提供了理论依据。

关键词: 高加索三叶草, 低温胁迫, 生理指标, 差异基因