苜蓿种子发芽特性和萌发早期抗氧化系统对老化的生理和分子响应研究

doi:10.11686/cyxb2024506

摘要/Abstract

摘要：

为研究老化苜蓿种子发芽特性、萌发早期抗氧化系统生理变化规律以及探索抗氧化相关基因对种子老化的响应模式，以老化紫花苜蓿种子为材料，研究了老化处理后种子发芽特性、萌发早期种子抗氧化酶活性和抗氧化物含量的变化规律，并分析了抗氧化系统相关基因表达量的变化。结果表明，老化处理不仅抑制了苜蓿种子萌发进程，同时显著影响了种子萌发后幼苗的正常生长，导致萌发后期幼苗较小。老化处理也严重影响了活性氧（ROS）的代谢，导致老化种子萌发早期过氧化氢（H₂O₂）含量升高，过氧化物酶（POD）和谷胱甘肽还原酶（GR）活性显著（P<0.05）降低，抗坏血酸（AsA）和谷胱甘肽（GSH）含量也极显著（P<0.01）降低。抗氧化酶活性和抗氧化物含量降低使种子抗氧化能力降低。此外，老化处理也显著（P<0.05）抑制了抗氧化系统中MsCAT1、MsPOD12、MsDHAR、MsGR1、MsFe-SOD和MsMn-SOD的表达。基于抗氧化生理和基因表达模式分析，从抗氧化系统中挖掘到一些关键的候选基因，为深入研究种子老化的分子调控机制提供了重要基因资源，也为进一步研究这些候选基因调控种子活力的精确途径奠定了基础。

关键词: 紫花苜蓿, 发芽特性, 萌发早期, 抗氧化酶, 抗氧化物, 分子响应

Abstract:

The aims of this study were to investigate the germination characteristics of aged alfalfa （Medicago sativa） seeds， the physiological changes in their antioxidant system during the early stage of germination， and the responses of antioxidant-related genes to seed aging. We observed seed germination characteristics and determined antioxidant enzyme activities and antioxidant contents during the early stage of germination following an aging treatment， and analyzed the transcript profiles of genes related to the antioxidant system. The results show that an aging treatment not only inhibited the germination of alfalfa seeds， but also significantly affected seedling growth after germination， leading to smaller seedlings. The aging treatment also severely impacted the metabolism of reactive oxygen species， resulting in increased H₂O₂ levels during early germination of aged seeds， a significant decrease in the activities of the antioxidant enzymes peroxidase and glutathione reductase， and a marked reduction in the contents of the antioxidants ascorbic acid and glutathione. The decrease in antioxidant enzyme activity and antioxidant content reduced the seeds’ antioxidant capacity. Additionally， the aging treatment led to significantly lower transcript levels of MsCAT1， MsPOD12， MsDHAR， MsGR1， MsFe-SOD， and MsMn-SOD，which encode important enzymes in the antioxidant system. On the basis of analyses of antioxidant physiology and gene transcript profiles， several key candidate genes in the antioxidant system were identified. These genes will be important targets for further research on the molecular regulation of seed aging. The results of this study lay the foundation for further studies on the precise pathways through which these candidate genes regulate seed vigor.

Key words: alfalfa, germination characteristics, early germination stage, antioxidant enzyme, antioxidant, molecular response

孙守江, 刘昊臻, 徐淑涵, 张景鈜, 李淑霞, 张金青, 高雪芹, 伏兵哲. 苜蓿种子发芽特性和萌发早期抗氧化系统对老化的生理和分子响应研究[J]. 草业学报, 2025, 34(11): 195-204.

Shou-jiang SUN, Hao-zhen LIU, Shu-han XU, Jing-hong ZHANG, Shu-xia LI, Jin-qing ZHANG, Xue-qin GAO, Bing-zhe FU. Germination characteristics and antioxidant responses of alfalfa seeds to aging treatment[J]. Acta Prataculturae Sinica, 2025, 34(11): 195-204.

图/表 6

表1 qRT-PCR分析引物信息

Table 1 Primers for qRT-PCR analysis

编号 No.	基因名称 Gene name	引物序列 Primer sequence （5′-3′）	产物大小 Product size （bp）	溶解温度 Melting temperature （℃）
1	MsCAT1	F： ACAGGGATGAGGAGGTGAACTAC R： CGCTGGATGAAACGGTCTTGC	130	63.6 63.3
2	MsPOD12	F： CCCAAGGCAGAGTCCATCGTG R： CCGTCCAGGAGCACTGAAGC	102	60.0 59.8
3	MsAPX	F： AAGGACTATGCTGAATCACACAAG R： CAACTGCTACGCCGACTGC	111	56.9 58.5
4	MsDHAR	F： AAGACCAAGCCAGCCAAGGAG R： AAGGACAGGACAGAACACAGACG	96	60.2 60.9
5	MsCu/Zn-SOD	F： ACCGCCGTGACAGGAAGC R： GCAACACCATCCGCTCCAG	85	60.7 60.5
6	MsFe-SOD	F： AGAAGCACCACGCCACCTAC R： CGCCGCCGTTGAACTTGATG	112	59.1 59.8
7	MsMn-SOD	F： GCTGATGTGCTTGAATCCTTGAAC R： GTGCTTGTGCTGGTGAACTCC	147	59.7 59.0
8	MsGR1	F： AGAACAGCATATCCAAACGACAG R： CAGCCTTGAGTGCGACAGC	133	60.0 59.7
9	MsAOX1c	F： GCTGGAGGAGGCGGAGAAC R： TCGGTGTAGGAGTGGATGGC	96	58.4 56.5
10	MsNAC67	F： TGCCAAGCCTGCCCTGATATG R： ATTATTGTTGCCCACCTCGTTTCC	142	58.8 59.7
11	MsNAC74	F： CCGCCTTGCCGACACCTC R： TCTTCTCCCATTCGTTCTTCTTGC	101	58.6 59.9
12	MsNAC83	F： CGGAAGGCTGGGTGCTCTG R： TCGTGGACGGGATCTTCTTGG	124	60.3 60.1
13	MsACTIN	F： CAAAAGATGGCAGATGCTGAGGAT R： CATGACACCAGTATGACGAGGTCG	88	59.4 59.5

图1 老化苜蓿种子萌发特性的变化*表示在0.05水平下差异显著* indicate significant differences at the 0.05 level. CK：未老化Unaged； Aged：老化10 d Aged 10 days. 下同The same below.

Fig.1 Changes in germination related indicators of aged alfalfa seeds

图2 老化苜蓿种子萌发早期抗氧化酶活性的变化**： P<0.01； ***： P<0.001. 下同The same below.

Fig.2 Changes in antioxidant enzyme activities during the early germination stage of aged alfalfa seeds

图3 老化苜蓿种子萌发早期抗氧化物含量的变化ns表示无显著差异。ns indicate no significant differences.

Fig.3 Changes in antioxidant content during the early germination stage of aged alfalfa seeds

图4 老化苜蓿种子抗氧化系统和ROS代谢相关基因表达量的变化

Fig.4 Changes in the expression levels of genes related to the antioxidant system and ROS metabolism of aged alfalfa seeds

图5 苜蓿种子抗氧化系统对老化处理的响应示意图H2O2：过氧化氢Hydrogen peroxide； O2?-：超氧阴离子Superoxide anion； SOD：超氧化物歧化酶Superoxide dismutase； CAT：过氧化氢酶Catalase； POD：过氧化物酶Peroxidase； APX：抗坏血酸过氧化物酶Ascorbate peroxidase； GR：谷胱甘肽还原酶Glutathione reductase； DHAR：去氢抗坏血酸还原酶Dehydroascorbate reductase； MDHAR：单脱氢抗坏血酸还原酶Monodehydroascorbate reductase； AsA：抗坏血酸Ascorbic acid； DHA：去氢抗坏血酸Dehydroascorbic acid； GSH：谷胱甘肽Glutathione； GSSG：氧化型谷胱甘肽Glutathione oxidized； NADPH：还原型辅酶Ⅱ Reduced nicotinamide adenine dinucleotide phosphate； MDHA：单脱氢抗坏血酸Monodehydroascorbic acid； NADPH：还原型烟酰胺腺嘌呤二核苷酸Reduced nicotinamide adenine dinucleotide phosphate.

Fig.5 Schematic diagram of the response of the antioxidant system in alfalfa seeds to ageing treatments

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