Acta Agrestia Sinica

Acta Agrestia Sinica ›› 2024, Vol. 32 ›› Issue (7): 2028-2038.DOI: 10.11733/j.issn.1007-0435.2024.07.004

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Screening and Validation of Reference Genes for Xanthopappus subacaulis by Real-time Quantitative PCR under Abiotic Stress

YU Ming-jun1,2, SU Dan-dan1,2, SU Xu1,2,3, CHEN Jin-yuan1,2, LIU Yu-ping1,2, ZHANG Yu1,2, LIU Tao4, YANG Qian1,2, NIU Fu-ying1   

  1. 1. School of Life Sciences, Qinghai Normal University, Xining, Qinghai Province 810008, China;
    2. Key Laboratory of Biodiversity Formation Mechanism and Comprehensive Utilization of the Qinghai-Tibet Plateau in Qinghai Province, Qinghai Normal University, Xining, Qinghai Province 810008, China;
    3. Academy of Plateau Science and Sustainability, Qinghai Normal University, Xining, Qinghai Province 810016, China;
    4. School of Ecology and Environmental Science, Qinghai University of Science and Technology, Xining, Qinghai Province 810016, China
  • Received:2023-11-24 Revised:2024-03-26 Published:2024-08-03

非生物胁迫下黄缨菊实时荧光定量PCR内参基因的筛选与验证

余明君1,2, 苏丹丹1,2, 苏旭1,2,3, 陈金元1,2, 刘玉萍1,2, 张雨1,2, 刘涛4, 杨倩1,2, 牛富英1   

  1. 1. 青海师范大学生命科学学院, 青海 西宁 810008;
    2. 青海师范大学青海省青藏高原生物多样性形成机制与综合利用重点实验室, 青海 西宁 810008;
    3. 青海师范大学高原科学与可持续发展研究院, 青海 西宁 810016;
    4. 青海理工大学生态与环境科学学院, 青海 西宁 810016
  • 通讯作者: 刘玉萍，E-mail:lyp8527970@126.com
  • 作者简介:余明君(2001-)，女，汉族，山西朔州人，硕士研究生，主要从事高山植物遗传多样性与分子进化研究，E-mail：15234934641@139.com
  • 基金资助:
    第二次青藏高原综合科学考察研究项目(2019QZKK0502)；青海理工大学(筹)“昆仑英才”人才引进科研项目(2023-QLGKLYCZX-012)资助

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Abstract: To screen the steadily expressed reference genes in the gene analysis,we analyzed the reference genes that were stable expression in Xanthopappus subacaulis. We used the root,stem and leaf of Xanthopappus subacaulis as experimental materials under different abiotic stressed,and screened eight candidate internal reference genes based on the full-length transcript sequencing. Meanwhile,we utilized qRT-PCR technology and combined geNorm,NormFinder,BestKeeper,and RefFinder software to comprehensively evaluate the expression stability of candidate reference genes. The results showed that SAND,TIP and GAPDH were the most stable reference genes in root,stem and leaf of X. subacaulis under cold stress,respectively. SAND,TUA and UBQ were the most stable in roots,stems and leaves under drought stress,respectively. RPS,RPS and SAND were the most stable in roots,stems and leaves under salt stress. In addition,we chose two genes with the best stability and one gene with the worst stability in the roots,stems and leaves as internal reference genes under drought treatment to evaluate the accuracy of expression level of the stress-tolerant gene WRKY,the results indicated that the stability of internal reference genes in different tissues of X. subacaulis were reliable under different stresses. The study provides the gene resources and theoretical basis for the expression analysis of stress-tolerant genes of X. subacaulis in the future.

Key words: Xanthopappus subacaulis, Reference gene, qRT-PCR, Abiotic stress, Expression stability

摘要： 为了筛选基因分析中黄缨菊(Xanthopappus subacaulis)稳定表达的内参基因，本研究以冷、盐和干旱胁迫下黄缨菊的根、茎和叶为材料，依据三代转录组数据选取8个常用内参基因(18S rRNA，GAPDH，PAL，UBQ，TIP，RPS，SAND，TUA)作为候选基因，利用qRT-PCR技术并结合geNorm,NormFinder,BestKeeper和RefFinder软件综合评价候选内参基因的表达稳定性。结果表明，冷胁迫下黄缨菊根、茎、叶中最佳稳定性的内参基因分别为SAND,TIP和GAPDH；干旱胁迫下最稳定的内参基因分别是SAND,TUA和UBQ；盐胁迫下稳定性最好的内参基因分别为RPS,RPS和SAND。此外，分别以干旱胁迫处理下黄缨菊根、茎、叶中2个稳定性最佳和1个稳定性最差的基因作为内参基因，衡量耐逆基因WRKY表达量的准确性，表明不同胁迫下筛选出的黄缨菊不同组织中内参基因的稳定性结果均是可靠的。研究结果为今后黄缨菊耐逆基因的表达分析提供了基因资源和理论依据。

关键词: 黄缨菊, 内参基因, 实时荧光定量PCR, 非生物胁迫, 表达稳定性

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