Evaluation and Screening of Reference Genes in Alfalfa （Medicago sativa L. ‘Gongnong No.1’） Different Tissues under Waterlogging Stress

doi:10.11733/j.issn.1007-0435.2026.04.002

Acta Agrestia Sinica ›› 2026, Vol. 34 ›› Issue (4): 1146-1157.DOI: 10.11733/j.issn.1007-0435.2026.04.002

Evaluation and Screening of Reference Genes in Alfalfa （Medicago sativa L. ‘Gongnong No.1’） Different Tissues under Waterlogging Stress

XU Yi^1,2, WANG Ting¹, GUO Yu-peng³, MIAO Xing-fen², ZHENG Xia^1,4, SHI Peng-jun^1,4

1. Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, Changsha, Hunan Province 410221, China;
2. College of Agriculture, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang Province 163319, China;
3. The College of Ecological Environment and Resources, Qinghai Minzu University, Xining, Qinghai Province 810007, China;
4. National Nanfan Research Institute, Chinese Academy of Agricultural Sciences, Sanya, Hainan Province 572024, China

Received:2025-04-15 Revised:2025-06-13 Published:2026-04-15

‘公农1号’苜蓿不同组织和涝害胁迫下内参基因的筛选评价

徐艺^1,2, 王婷¹, 郭玉朋³, 苗兴芬², 郑霞^1,4, 石鹏君^1,4

1. 中国农业科学院麻类研究所, 湖南长沙 410221;
2. 黑龙江八一农垦大学农学院, 黑龙江大庆 163319;
3. 青海民族大学生态环境与资源学院, 青海西宁 810007;
4. 中国农业科学院国家南繁研究院, 海南三亚 572024

通讯作者: 苗兴芬，E-mail：byndmxf@126.com；郑霞，E-mail：zhengxia@caas.cn
作者简介:徐艺（1996-），女，汉族，山东济宁人，硕士研究生，主要从事牧草种质资源创新与利用研究，E-mail：xuyi01010219@163.com；王婷（1998-），女，汉族，甘肃会宁人，硕士研究生，主要从事苜蓿逆境分子生物学研究，E-mail：82101235179@caas.cn；
基金资助:
湖南省自然科学基金-省市联合基金（2022JJ50021）；中国农业科学院科技创新工程（CAAS-ASTIP-IBFC）；三亚中国农业科学院国家南繁研究院“南繁专项”（YYLH2505）资助

Abstract

Abstract: The stability and applicability of reference genes in alfalfa vary significantly depending on the cultivar， growth and development stages， and environmental conditions. In this study， alfalfa （Medicago sativa L.） cultivar ‘Gongnong No.1’ was used as material， and eight candidate reference genes， glyceraldehyde-3-phosphate dehydrogenase GAPDH， ubiquitin protein ligase 2a UBL-2a， Retention in Endoplasmic Reticulum Rer1， eukaryotic elongation factor-1 alpha eEF-1α， actin-depolymerizing factor ADF， ubiquitin carrier protein UBC， tubulin beta-1 chain TUB， and Actin were selected to evaluate their expression stability of different tissues in seedling and adult plant， as well as under waterlogging stress. The analysis employed quantitative real-time PCR （qRT-PCR） and four softwares， including geNorm， NormFinder， BestKeeper， and RefFinder. The results showed that， at the seedling stage， Rer1， Actin， and UBC were the most stable reference genes. At the tissue level， the most stable reference genes for leaves， stems， and roots were Actin， Rer1， and GAPDH， respectively. At the adult stage， ADF， Actin， and TUB exhibited the highest stability， while the most stable reference genes for leaves， stems， roots， and flowers were eEF-1α， Rer1， Actin， and UBL-2a， respectively. Under waterlogging stress， the three most stable reference genes in seedling leaves were TUB， ADF， and eEF-1α， whereas in seedling roots they were ADF， UBL-2a， and Rer1. In the adult stage， the most stable genes were ADF， Actin， and TUB in leaves， and UBL-2a， UBC， and ADF in roots. Overall， Rer1 and UBL-2a are recommended as reference genes for studies of tissue-specific expression， as their stability was less affected by developmental stage or tissue type. ADF and TUB are suitable reference genes for studies involving waterlogging stress.

Key words: Alfalfa, Reference genes, Abiotic stress, Expression stability

摘要： 紫花苜蓿（Medicago sativa L.）内参基因的稳定性和适用性因品种、生长发育及环境条件的不同差异较大。本研究以紫花苜蓿‘公农1号’为材料，使用qRT-PCR技术与geNorm，NormFinder，BestKeeper和RefFinder等软件，评估8个常见内参基因GAPDH，UBL-2a，Rer1，eEF-1α，ADF，UBC，TUB和Actin等不同时期的组织表达特异性以及涝害胁迫下的表达稳定性。结果表明，‘公农1号’幼苗期稳定的内参基因是Rer1，Actin，UBC，其中叶、茎和根中表达最稳定的内参基因分别是Actin，Rer1，GAPDH；成株期最稳定的内参基因是ADF，Actin，TUB，而成株期叶、茎、根和花中表达最稳定的分别是eEF-1α，Rer1，Actin和UBL-2a。涝害胁迫下，幼苗期叶片稳定性前三的内参基因为TUB，ADF和eEF-1α，根部为ADF，UBL-2a和Rer1；成株期叶中稳定性前三的内参基因为ADF，Actin和TUB，根部为UBL-2a，UBC和ADF。Rer1和UBL-2a的稳定性受发育时期和组织特异性影响较小，适合用作组织表达特异性研究的内参基因；ADF适合作为涝害胁迫研究的内参基因。

关键词: 紫花苜蓿, 内参基因, 非生物胁迫, 表达稳定性

CLC Number:

S541

XU Yi, WANG Ting, GUO Yu-peng, MIAO Xing-fen, ZHENG Xia, SHI Peng-jun. Evaluation and Screening of Reference Genes in Alfalfa （Medicago sativa L. ‘Gongnong No.1’） Different Tissues under Waterlogging Stress[J]. Acta Agrestia Sinica, 2026, 34(4): 1146-1157.

徐艺, 王婷, 郭玉朋, 苗兴芬, 郑霞, 石鹏君. ‘公农1号’苜蓿不同组织和涝害胁迫下内参基因的筛选评价[J]. 草地学报, 2026, 34(4): 1146-1157.

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Evaluation and Screening of Reference Genes in Alfalfa （Medicago sativa L. ‘Gongnong No.1’） Different Tissues under Waterlogging Stress

‘公农1号’苜蓿不同组织和涝害胁迫下内参基因的筛选评价

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