梭梭qRT-PCR内参基因的筛选及稳定性分析
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西北农林科技大学林学院

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国家自然科学基金项目(31960333);中央高校基本科研业务费(2452023021);新疆维吾尔自治区自然科学基金项目(2019D01C017)


Screening and stability analysis of internal reference genes for qRT-PCR in Haloxylon ammodendron
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    摘要:

    本研究旨在探明梭梭(Haloxylon ammodendron)在不同非生物胁迫下稳定表达的内参基因,为后续梭梭抗逆性相关基因功能研究奠定基础。研究采用实时定量聚合酶链式反应(qRT-PCR)技术从梭梭转录组数据库中检测了GAPDH、ef1-α、UBC、RPL32、ALB、50S-1721、50S-1063、RPⅡ、H3、PP2A、SOD、HSC70、TUA和TUB等14个候选内参基因在高温、干旱、盐、ABA和昼夜节律条件下的表达变化。利用geNorm、NormFinder、BestKeeper和RefFinder软件对梭梭候选内参基因的稳定性进行评价,最终筛选出合适的内参基因,并通过对梭梭磷酸烯醇丙酮酸羧化酶(phosphoenolpyruvate carboxylase, PEPC)基因表达分析,验证了不同内参基因对实验结果的影响。4种软件分析得到的最优内参基因存在差异,在RefFinder网站上综合排序分析表明,在ABA处理和昼夜节律下,ALB是最优内参基因,RPⅡ基因在干旱胁迫下表达最稳定,TUB和RPⅡ基因在盐胁迫下最适用,H3基因在高温胁迫下表达最为稳定。各种胁迫下最适宜的内参基因为ALB和RPL32。通过计算几何平均值,得到14个候选内参基因的综合稳定性排名,其中排名前两位的基因分别为SOD和RPL32。综上,RPL32和SOD可作为梭梭qRT-PCR标准化的内参基因。

    Abstract:

    The aim of this study is to find out the reference genes stably expressed in various abiotic stresses of Haloxylon ammodendron, so as to lay a foundation for the subsequent study on the function of genes related to stress resistance of H. ammodendron. In this study, real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression changes of 14 candidate internal reference genes, including GAPDH, EF-1α, UBC, RPL32, ALB, 50S-1721, 50S-1063, RPⅡ, H3, PP2A, SOD, HSC70, TUA, and TUB, from the transcriptome database of H. ammodendron, under high temperature, drought, salt, abscisic acid (ABA) and circadian rhythms. By using geNorm, NormFinder, BestKeeper and RefFinder software, the stability of candidate internal reference genes of H. ammodendron was evaluated, and suitable internal reference genes were finally selected. Furthermore, the expression of phosphoenolpyruvate carboxylase (PEPC) gene was analyzed to verify the influence of different reference genes on the experimental results. And significant differences were found in the optimal internal reference genes analyzed by the four software programs, and the comprehensive ranking analysis on the RefFinder website showed that ALB was the optimal internal reference gene under ABA treatment and circadian rhythms, the RPⅡ gene was most stably expressed in leaves under drought stress, TUB and RPⅡ were most suitable internal reference genes under salt stress, and the H3 gene was most stably expressed under high temperature stress. The most suitable internal reference genes under all stresses were ALB and RPL32. By calculating the geometric mean, the comprehensive stability ranking of 14 candidate internal reference genes was obtained, of which the top two genes were SOD and RPL32, respectively. In conclusion, RPL32 and SOD can be used as internal reference genes for qRT-PCR standardization.

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张玲玲,王淑冉,张胜.梭梭qRT-PCR内参基因的筛选及稳定性分析[J].西北植物学报,2023,43(12):2005-2017

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  • 收稿日期:2023-06-04
  • 最后修改日期:2023-11-09
  • 录用日期:2023-11-24
  • 在线发布日期: 2023-12-04
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