EF1α and αTUB Are Stable Reference Gene Pairs for RT-qPCR-Based Gene Expression Studies in Salix suchowensis Under Nitrogen Treatment Conditions
Abstract
1. Introduction
2. Results
2.1. Verification of Primer Specificity and Amplification Efficiency
2.2. Expression Profiles of Reference Genes in Roots, Stems, and Leaves Under N Treatments
2.3. Expression Stability Analysis of the Selected Eight Candidate Reference Genes
2.4. Validation of the Stability of the Selected Candidate Reference Genes
3. Discussion
4. Materials and Methods
4.1. Growth of Salix suchowensis and Harvest
4.2. RNA Extraction, First-Strand cDNA Synthesis, and RT-qPCR Analysis
4.3. Selection of Candidate Reference Genes and Primer Design
4.4. Establishment of Reference Gene Primer Standard Curve
4.5. Statistical Analysis
4.6. Gene Expression Level Analysis Using Various Reference Genes
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
References
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Gene | Locus | Protein Size (aa) | Gene Description | Gene Structure |
---|---|---|---|---|
EF1α | KAG5219160.1 | 229 | elongation factor 1 alpha | |
EFβ | KAG5253579.1 | 223 | elongation factor beta | |
αTUB | KAG5254066.1 | 451 | alpha−tubulin | |
βTUB | KAG5249312.1 | 446 | beta−tubulin | |
GAPDH | KAG5246615.1 | 453 | glyceraldehyde−3−phosphate dehydrogenase | |
18s | KAG5248457.1 | 289 | 18S rRNA (guanine−N(7)) −methyltransferase | |
Actin1 | KAG5252779.1 | 267 | actin | |
Actin2 | KAG5255349.1 | 257 | actin | |
H2A1 | KAG5232433.1 | 144 | histone H2A | |
H2A2 | KAG5246854.1 | 130 | histone H2A | |
UBQ1 | KAG5248096.1 | 135 | ubiquitin | |
UBQ3 | KAG5221099.1 | 186 | ubiquitin | |
H2B2 | KAG5239632.1 | 139 | histone H2B | |
Rank | 1/2 | 3 | 4 | 5 | 6 | 7 | 8 | ||
---|---|---|---|---|---|---|---|---|---|
(Gene)Stability | |||||||||
Treatments | NH4NO3 | (EF1α/EFβ) 0.25 | (αTUB) 0.34 | (βTUB) 0.52 | (H2A1) 0.74 | (Actin2) 0.91 | (Actin1) 1.14 | (GAPDH) 1.62 | |
NH4+ | (EF1α/EFβ) 0.33 | (αTUB) 0.45 | (Actin2) 0.56 | (βTUB) 0.71 | (Actin1) 0.95 | (H2A1) 1.37 | (GAPDH) 1.70 | ||
NO3− | (αTUB/βTUB) 0.20 | (EFβ) 0.39 | (EF1α) 0.50 | (Actin2) 0.79 | (Actin1) 1.02 | (H2A1) 1.21 | (GAPDH) 1.73 | ||
−N | (EF1α/EFβ) 0.35 | (αTUB) 0.42 | (βTUB) 0.53 | (Actin2) 0.68 | (H2A1) 0.88 | (Actin1) 1.10 | (GAPDH) 1.50 | ||
Tissues | Root | (EF1α/βTUB) 0.18 | (EFβ) 0.19 | (αTUB) 0.24 | (ACT1) 0.38 | (ACT2) 0.57 | (H2A1) 0.70 | (GAPDH) 0.83 | |
Stem | (EF1α/αTUB) 0.29 | (EFβ) 0.32 | (Actin1) 0.35 | (βTUB) 0.39 | (GAPDH) 0.49 | (ACT2) 0.66 | (H21A) 0.83 | ||
Leaf | (αTUB/GAPDH) 0.23 | (EF1α) 0.26 | (EFβ) 0.28 | (βTUB) 0.33 | (Actin1) 0.38 | (Actin2) 0.44 | (H2A1) 0.87 | ||
Total | (EFβ/EF1α) 0.35 | (αTUB) 0.44 | (βTUB) 0.59 | (Actin2) 0.80 | (Actin1) 1.04 | (H2A1) 1.26 | (GAPDH) 1.68 |
Rank | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | ||
---|---|---|---|---|---|---|---|---|---|---|
(Gene)Stability | ||||||||||
Treatments | NH4NO3 | (αTUB) 0.123 | (EFβ) 0.176 | (βTUB) 0.268 | (EF1α) 0.348 | (H2A1) 0.544 | (Actin2) 0.620 | (Actin1) 1.321 | (GAPDH) 2.062 | |
NH4+ | (EFβ) 0.115 | (αTUB) 0.172 | (Actin2) 0.199 | (EF1α) 0.275 | (βTUB) 0.518 | (Actin1) 1.034 | (H2A1) 1.669 | (GAPDH) 1.739 | ||
NO3− | (αTUB) 0.069 | (βTUB) 0.069 | (EFβ) 0.145 | (EF1α) 0.432 | (Actin2) 0.560 | (H2A1) 0.955 | (Actin1) 1.273 | (GAPDH) 2.203 | ||
−N | (EFβ) 0.082 | (αTUB) 0.143 | (βTUB) 0.154 | (EF1α) 0.251 | (Actin2) 0.398 | (H2A1) 0.824 | (Actin1) 1.254 | (GAPDH) 1.804 | ||
Tissues | Root | (αTUB) 0.173 | (EFβ) 0.210 | (βTUB) 0.217 | (EF1α) 0.231 | (Actin1) 0.488 | (Actin2) 0.502 | (H2A1) 0.602 | (GAPDH) 0.748 | |
Stem | (EF1α) 0.192 | (EFβ) 0.234 | (αTUB) 0.316 | (Actin1) 0.316 | (βTUB) 0.335 | (GAPDH) 0.374 | (Actin2) 0.570 | (H2A1) 0.879 | ||
Leaf | (αTUB) 0.078 | (GAPDH) 0.078 | (EFβ) 0.174 | (EF1α) 0.241 | (Actin1) 0.254 | (βTUB) 0.292 | (Actin2) 0.371 | (H2A1) 1.463 | ||
Total | (EFβ) 0.041 | (αTUB) 0.153 | (EF1α) 0.334 | (βTUB) 0.357 | (Actin2) 0.500 | (H2A1) 1.095 | (Actin1) 1.212 | (GAPDH) 1.951 |
Rank | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | ||
---|---|---|---|---|---|---|---|---|---|---|
Treatments | NH4NO3 | Gene | EF1α | EFβ | αTUB | H2A1 | βTUB | Actin2 | Actin1 | GAPDH |
SD/CV | 0.66/3.23 | 0.66/2.74 | 0.71/2.97 | 0.79/2.87 | 0.89/3.39 | 1.27/4.49 | 1.82/6.59 | 2.05/8.27 | ||
NH4+ | Gene | Actin2 | EFβ | EF1α | αTUB | βTUB | GAPDH | H2A1 | Actin1 | |
SD/CV | 1.07/3.80 | 1.08/4.34 | 1.20/5.72 | 1.23/4.99 | 1.30/4.89 | 1.55/6.34 | 1.86/6.56 | 2.01/7.06 | ||
NO3− | Gene | EF1α | EFβ | H2A1 | αTUB | βTUB | Actin2 | Actin1 | GAPDH | |
SD/CV | 0.67/3.30 | 0.71/2.96 | 0.72/2.64 | 0.91/3.83 | 1.05/4.08 | 1.15/4.07 | 1.65/6.01 | 2.72/11.01 | ||
−N | Gene | EFβ | EF1α | αTUB | βTUB | Actin2 | H2A1 | GAPDH | Actin1 | |
SD/CV | 0.71/2.84 | 0.74/3.57 | 0.89/3.65 | 0.96/3.56 | 1.10/3.88 | 1.10/4.07 | 1.84/7.48 | 1.86/6.67 | ||
Tissues | Root | Gene | αTUB | Actin1 | EF1α | βTUB | EFβ | H2A1 | Actin2 | GAPDH |
SD/CV | 0.43/1.79 | 0.43/1.59 | 0.44/2.19 | 0.46/1.75 | 0.49/2.05 | 0.84/3.14 | 0.88/3.03 | 1.16/4.29 | ||
Stem | Gene | H2A1 | GAPDH | Actin2 | βTUB | EF1α | EFβ | αTUB | Actin1 | |
SD/CV | 0.73/2.66 | 0.87/3.48 | 0.94/3.46 | 0.94/3.75 | 0.96/4.65 | 0.96/3.95 | 1.05/4.47 | 1.11/4.24 | ||
Leaf | Gene | EF1α | αTUB | Actin1 | EFβ | GAPDH | Actin2 | βTUB | H2A1 | |
SD/CV | 0.48/2.25 | 0.63/2.55 | 0.64/2.10 | 0.69/2.75 | 0.70/3.22 | 0.77/2.68 | 0.78/2.86 | 1.64/5.86 | ||
Total | Gene | EFβ | EF1α | αTUB | βTUB | Actin2 | H2A1 | Actin1 | GAPDH | |
SD/CV | 0.80/3.26 | 0.83/4.03 | 0.92/3.81 | 1.07/4.05 | 1.14/4.04 | 1.15/4.19 | 1.85/6.64 | 2.07/8.39 |
Rank | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | ||
---|---|---|---|---|---|---|---|---|---|---|
Treatments | NH4NO3 | Gene | αTUB | EFβ | βTUB | EF1α | H2A1 | Actin2 | Actin1 | GAPDH |
NH4+ | Gene | EFβ | αTUB | Actin2 | EF1α | βTUB | Actin1 | H2A1 | GAPDH | |
NO3− | Gene | αTUB | βTUB | EFβ | EF1α | Actin2 | H2A1 | Actin1 | GAPDH | |
−N | Gene | EFβ | αTUB | βTUB | EF1α | Actin2 | H2A1 | Actin1 | GAPDH | |
Tissues | Root | Gene | αTUB | EFβ | βTUB | EF1α | Actin1 | Actin2 | H2A1 | GAPDH |
Stem | Gene | EF1α | EFβ | αTUB | Actin1 | βTUB | GAPDH | Actin2 | H2A1 | |
Leaf | Gene | αTUB | GAPDH | EFβ | EF1α | Actin1 | βTUB | Actin2 | H2A1 | |
Total | Gene | EFβ | αTUB | EF1α | βTUB | Actin2 | H2A1 | Actin1 | GAPDH |
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Huang, L.; Zhang, Y.; Gao, F.; Fu, Y.; Sun, J.; Zhou, J.; Tao, J.; He, X.; Guo, N. EF1α and αTUB Are Stable Reference Gene Pairs for RT-qPCR-Based Gene Expression Studies in Salix suchowensis Under Nitrogen Treatment Conditions. Plants 2025, 14, 3101. https://doi.org/10.3390/plants14193101
Huang L, Zhang Y, Gao F, Fu Y, Sun J, Zhou J, Tao J, He X, Guo N. EF1α and αTUB Are Stable Reference Gene Pairs for RT-qPCR-Based Gene Expression Studies in Salix suchowensis Under Nitrogen Treatment Conditions. Plants. 2025; 14(19):3101. https://doi.org/10.3390/plants14193101
Chicago/Turabian StyleHuang, Lei, Yuyi Zhang, Fei Gao, Yu Fu, Jing Sun, Jie Zhou, Jun Tao, Xudong He, and Nan Guo. 2025. "EF1α and αTUB Are Stable Reference Gene Pairs for RT-qPCR-Based Gene Expression Studies in Salix suchowensis Under Nitrogen Treatment Conditions" Plants 14, no. 19: 3101. https://doi.org/10.3390/plants14193101
APA StyleHuang, L., Zhang, Y., Gao, F., Fu, Y., Sun, J., Zhou, J., Tao, J., He, X., & Guo, N. (2025). EF1α and αTUB Are Stable Reference Gene Pairs for RT-qPCR-Based Gene Expression Studies in Salix suchowensis Under Nitrogen Treatment Conditions. Plants, 14(19), 3101. https://doi.org/10.3390/plants14193101