| 舒本水，黄玉婷，余萱悦，刘翠婷，谢心怡，沈 皓，林进添.印楝素胁迫下草地贪夜蛾幼虫实时荧光定量 PCR 内参基因表达稳定性评价[J].广东农业科学,2024,51(8):21-30 |
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印楝素胁迫下草地贪夜蛾幼虫实时荧光定量 PCR 内参基因表达稳定性评价 |
| Expression Stability of Reference Genes in Larvae of Spodoptera frugipferda Under Azadirachtin Stress by Real-Time Quantitative PCR Analysis |
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| DOI：10.16768/j.issn.1004-874X.2024.08.003 |
| 中文关键词: 实时荧光定量 PCR 草地贪夜蛾 印楝素 内参基因 表达稳定性 |
| 英文关键词: real-time quantitative PCR Spodoptera frugiperda azadirachtin reference gene expression stability |
| 基金项目:国家自然科学基金（32102221）； 广东省重点领域研发计划项目（2020B020223004）；广东省大学生
创新创业训练计划项目（S202311347039） |
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| 摘要点击次数: 188 |
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| 中文摘要: |
| 【目的】选择合适的内参基因对实时荧光定量 PCR 技术结果的准确性具有重要意义。通过评估印楝
素胁迫下草地贪夜蛾（Spodoptera frugiperda J.E Smith）幼虫候选内参基因在不同样品中的表达稳定性，确定最适
内参基因，为后续靶标基因功能分析和印楝素分子毒理机制解析提供基础。【方法】选取草地贪夜蛾 α-TUB、β-1-TUB、
Actin、EF1α、EF2、GAPDH、RPL3 和 RPL13 8 个候选内参基因，通过 ΔCt、GeNorm、NormFinder、BestKeeper 和
RefFinder 软件对印楝素胁迫下各候选内参基因在草地贪夜蛾幼虫整虫、表皮、中肠、脂肪体和马氏管样品中转录
水平表达量的稳定性进行评估，采用 GeNorm 软件确定最适内参基因数目，最终确定印楝素胁迫下不同样品中最适
内参基因组合。【结果】印楝素胁迫下草地贪夜蛾幼虫样品中，ΔCt 和 Normfinder 分析发现 α-TUB 和 β-1-TUB 的表
达稳定性最高，GeNorm 分析发现 RPL3 和 RPL13 的表达稳定性最高，BestKeeper 分析则是 α-TUB 和 RPL13 表达稳
定性最高。表皮样品中，β-1-TUB 和 α-TUB 在 ΔCt、GeNorm 和 Normfinder 中表达稳定性最高，Actin 和 β-1-TUB 在
BestKeeper 评估中最稳定。在中肠组织中，ΔCt 分析发现 EF2 和 β-1-TUB 表达最稳定，GeNorm 发现 EF1α 和 RPL3
表达最稳定，Normfinder 分析发现 EF2 和 β-1-TUB 表达最稳定，BestKeeper 分析发现 RPL13 和 RPL3 表达最稳定。
在脂肪体组织中，ΔCt、GeNorm 和 Normfinder 分析下 RPL3 和 EF1α 表达稳定性最高，BestKeeper 分析则认为 RPL13
和 RPL3 稳定性最高。在马氏管组织中，ΔCt 分析发现 RPL3 和 EF2 表达最稳定，GeNorm 评估 EF1α 和 EF2 表达最
稳定，Normfinder 分析发现 β-1-TUB 和 EF2 表达最稳定，BestKeeper 分析发现 RPL13 和 β-1-TUB 表达最稳定。此外，
GeNorm 软件分析发现各样品中最适内参基因数目均为 2 个。RefFinder 综合分析结果为，α-TUB 和 β-1-TUB 在幼虫
和表皮组织样品中表达稳定性最高；EF2 和 RPL3 在幼虫中肠和马氏管组织样品中表达稳定性最高；RPL3 和 EF1α
在脂肪体组织样品中表达稳定性最高。【结论】印楝素胁迫下草地贪夜蛾幼虫和表皮中最适内参基因组合为 α-TUB
和 β-1-TUB；幼虫中肠和马氏管组织样品中最适内参基因组合为 EF2 和 RPL3；脂肪体组织样品中最适内参基因组
合为 RPL3 和 EF1α。 |
| 英文摘要: |
| 【Objective】Real-time quantitative PCR is one of the most conventional and efficient techniques for studying
the mRNA expression level of target genes. The proper selection of reference genes is of great significance for the accuracy of
the results. The study aimed to identify the most suitable reference genes by evaluating the expression stability of candidate
reference genes in different samples of Spodoptera frugiperda larvae under azadirachtin stress. The results provided a basis
for functional analysis of target genes and molecular toxicological mechanism analysis of azadirachtin.【Method】 Eight
candidate reference genes, including α-TUB, β-1-TUB, Actin, EF1α, EF2, GAPDH, RPL3 and RPL3, of S. frugiperda were
selected. The expression stability of these genes in different samples of larvae, cuticle, midgut, fat body and malpighian tube
under azadirachtin stress was evaluated by the ΔCt, GeNorm, NormFinder, BestKeeper and RefFinder software. And the optimal
number of reference genes were determined by GeNorm software. Finally, the best reference gene combinations in different
samples were comfirmed.【Result】When exposed to azadirachtin stress, in larvae samples, the expression stability of α-TUB
and β-1-TUB was the highest by ΔCt and Normfinder analyses; the expression stability of RPL3 and RPL13 was the highest by
GeNorm analysis; and the expression stability of α-TUB and RPL13 was the highest by BestKeeper analysis. In cuticle samples,
β-1-TUB and α-TUB were rated as the most stable genes in ΔCt, GeNorm and Normfinder, while Actin and β-1-TUB were the
most stable in BestKeeper. In the midgut tissue, ΔCt analysis found that EF2 and β-1-TUB expression was the most stable;
GeNorm assessment showed that EF1α and RPL3 expression was the most stable; Normfinder considered that EF2 and β-1-
TUB expression was the most stable, and BestKeeper analysis found that RPL13 and RPL3 expression was the most stable.
ΔCt, GeNorm and Normfinder analyses showed that RPL3 and EF1α were the most stable in fat body tissue, while BestKeeper
analysis showed that RPL13 and RPL3 were the most stable. In malpighian tube tissue, ΔCt analysis found that RPL3 and EF2
expression was the most stable, GeNorm assessment indicated that EF1α and EF2 expression was the most stable; Normfinder
considered that β-1-TUB and EF2 expression was the most stable, and BestKeeper analysis found that RPL13 and β-1-
TUB expression was the most stable. In addition, GeNorm software analysis found that the optimal number of reference genes
in each sample was 2. Therefore, as assessed by RefFinder comprehensive analysis, α-TUB and β-1-TUB had the highest
expression stability in larvae and cuticle tissue samples; EF2 and RPL3 had the highest expression stability in larval midgut and
malpighian tube samples; and RPL3 and EF1α had the highest expression stability in fat body samples.【Conclusion】Under
azadirachtin stress, the optimal reference genes for different samples of S. frugiperda larvae were shown as: α-TUB and β-1-TUB
for larvae and cuticle samples; EF2 and RPL3 for midgut and malpighian tube samples; and RPL3 and EF1α for fat body samples. |
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