Selection and Validation of Reference Genes for Gene Expression Studies in Euonymus japonicus Based on RNA Sequencing
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plant Materials
2.2. RNA-Seq
2.3. RNA Extraction and cDNA Reverse Transcription
2.4. Primer Design and PCR
2.5. Data Analysis
3. Results
3.1. Screening of Candidate RGs Based on the Results of RNA-Seq
3.2. Primer Specificity and Amplification Efficiency of Candidate RGs
3.3. Expression Profiles of Candidate RGs
3.4. GeNorm Analysis
3.5. NormFinder Analysis
3.6. Bestkeeper Analysis
3.7. Comprehensive Analysis of RefFinder
3.8. Reference Gene Selection and Validation
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Category | Number | Gene | Gene Description | Homologous Sequence | Identities |
---|---|---|---|---|---|
Candidate traditional housekeeping genes | 1 | Actin7 | actin-7 | XM_038865290.1 | 96% |
2 | TuA | tubulin α-5 chain | XM_038840525.1 | 96% | |
3 | TuB | tubulin β chain-like | XM_038823732.1 | 95% | |
4 | eIF4A | eukaryotic initiation factor 4A-3 | XM_038845408.1 | 92% | |
5 | eIF5A | eukaryotic translation initiation factor 5A | XM_038856304.1 | 94% | |
6 | GAPDH | glyceraldehyde-3-phosphate dehydrogenase, cytosolic | XM_038838191.1 | 91% | |
7 | CYP72A | cytochrome P450 72A765 | MN738192.1 | 90% | |
8 | UBC2 | ubiquitin-conjugating enzyme E2 2 | XM_038844679.1 | 95% | |
9 | RAP2 | ethylene-responsive transcription factor RAP2-4 | XM_038860091.1 | 90% | |
10 | UBQ | polyubiquitin | XM_038834479.1 | 91% | |
11 | ClpA | ATP-dependent Clp protease ATP-binding subunit ClpA | XM_038830531.1 | 96% | |
12 | FtsH2 | ATP-dependent zinc metalloprotease FTSH 2 | XM_038853108.1 | 94% | |
Candidate new RGs | 1 | RTNLB1 | reticulon-like protein B1 | XM_038853888.1 | 94% |
2 | UNC | uncharacterized | XM_038867966.1 | 91% | |
3 | GAD | glutamate decarboxylase | XM_038864250.1 | 92% | |
4 | ALEU | thiol protease aleurain-like | XM_038855394.1 | 92% | |
5 | BLH1 | BEL1-like homeodomain protein 1 | XM_038829031.1 | 88% | |
6 | SBT3.17 | subtilisin-like protease SBT3.17 | XM_038863645.1 | 92% | |
7 | HMGB2 | high mobility group B protein 2-like | XM_038849099.1 | 92% | |
8 | GEPI48 | UDP-glucose 4-epimerase GEPI48-like | XM_038869380.1 | 96% | |
9 | SCPL | serine carboxypeptidase-like | XM_038834759.1 | 94% |
Category | Genes | Primer Sequence (5′-3′) Forward | Primer Sequence (5′-3′) Reverse | Product (bp *) | R2 * | E * (%) |
---|---|---|---|---|---|---|
Candidate traditional house-keeping genes | Actin7 | GTGTCATGGTTGGGATGGGT | ACGATACCGTGTTCGATGGG | 150 | 0.9975 | 103 |
TUA | ACTGGCTTCAAGTGCGGTAT | GATCGATGCGTCCAAACACC | 133 | 0.9962 | 104 | |
TUB | GCCGGACAATTTCGTCTTCG | TTCCTCACGACATCGAGCAC | 108 | 0.9988 | 104 | |
eIF4A | CCGGCAAGACCTCCATGATT | TTCCCTCGTAGGCGACACTA | 95 | 1.0000 | 108 | |
eIF5A | GAGGAGCACCACTTCGAGTC | GGGCGATTCTTGGTGACGAT | 130 | 0.9985 | 102 | |
GAPDH | GGCTTGAGAAGGAGGCTACC | ACCAGCCTTGGCGTCAAATA | 158 | 0.9991 | 110 | |
CYP72A | GACGCCGAGTGTGACGATAA | GGTCCATTTCTCGCCCTCAA | 142 | 0.9961 | 101 | |
UBC2 | GCTCTGGAACGCTGTCATTT | CGAAGCGAACTGTAGGAGGC | 116 | 0.9995 | 109 | |
RAP2 | GCTTTACCGAGGAGTCAGGC | CCTCGGCGGTATCAAAGGTA | 107 | 0.9921 | 105 | |
UBQ | CCCTTGAAGTGGAAAGCAGTG | ATCAGCCAGAGTCCTTCCATC | 134 | 0.9992 | 104 | |
ClpA | TGCTGGAACCAAGTACCGTG | TGCCCCAGCTCCAATTAAGG | 124 | 0.9990 | 105 | |
FtsH2 | GGAGCTGATCTTGCCAACCT | CCCTCCATTCCAGCCACAAT | 122 | 0.9967 | 104 | |
Candidate new RGs | RTNLB1 | CGGAGCATACTGGTGAGCAT | ATCGGAATCGGAAGACGACG | 104 | 0.9981 | 98 |
UNC | TGGTACTTCGGGTTTGCAGC | TTGATGGCGTGCGAAGGTAT | 135 | 0.9906 | 104 | |
GAD | TCAGTCCACTCCACTTTCGC | TTCCCGTCCAACATCAGCTC | 128 | 0.9988 | 110 | |
ALEU | GTCGGCAACACTCATAACGC | ACAGCGAGCTTGTAAGGCAA | 164 | 0.9989 | 105 | |
BLH1 | CCACCGCACTCCAACCTAAT | ATGAGTCCGTGCAAAGCAGA | 155 | 0.9933 | 104 | |
SBT3.17 | GAGGTTGACGCAATCGTTGT | ATGTGGACCTTCGATTCGGG | 99 | 0.9994 | 103 | |
HMGB2 | CCAAGGATCCGAACAAGCCT | CCAACAACGGCAACGGATTT | 115 | 0.9992 | 103 | |
GEPI48 | CTGGCATTGGTTGTGAGGTG | TTCACGTTCTGCCTTGTCCG | 173 | 0.9942 | 105 | |
SCPL | GTGGGCATTCCTGCTCTTCT | GACCAGACCACTTCATGGCA | 121 | 0.9968 | 103 |
Category | Rank | Leaves under Different Temperature Stresses | Leaves of Plants at Different Growth Stages | Different Organs | |||
---|---|---|---|---|---|---|---|
Gene | Stability Value | Gene | Stability Value | Gene | Stability Value | ||
Candidate traditional housekeeping genes | 1 | TUA | 0.25 | eIF5A | 0.135 | eIF5A | 0.308 |
2 | eIF5A | 0.335 | TUB | 0.2 | GAPDH | 0.438 | |
3 | CYP72A | 0.408 | GAPDH | 0.304 | ClpA | 0.573 | |
4 | UBC2 | 0.545 | FtsH2 | 0.4 | FtsH2 | 0.658 | |
5 | GAPDH | 0.603 | TUA | 0.411 | RAP2 | 0.745 | |
6 | ClpA | 0.635 | ClpA | 0.685 | TUA | 0.778 | |
7 | TUB | 0.642 | eIF4A | 0.689 | TUB | 0.796 | |
8 | eIF4A | 0.682 | CYP72A | 0.748 | Actin7 | 0.842 | |
9 | Actin7 | 1.071 | Actin7 | 0.778 | eIF4A | 0.924 | |
10 | UBQ | 1.231 | UBQ | 0.824 | UBQ | 1.019 | |
11 | FtsH2 | 1.389 | RAP2 | 0.943 | CYP72A | 1.245 | |
12 | RAP2 | 1.418 | UBC2 | 1.038 | UBC2 | 1.301 | |
Candidate new RGs | 1 | SCPL | 0.102 | ALEU | 0.237 | HMGB2 | 0.508 |
2 | RTNLB1 | 0.217 | HMGB2 | 0.249 | RTNLB1 | 0.518 | |
3 | SBT3.17 | 0.292 | SCPL | 0.433 | UNC | 0.535 | |
4 | GEPI48 | 0.373 | RTNLB1 | 0.451 | GAD | 0.608 | |
5 | UNC | 0.467 | UNC | 0.507 | BLH1 | 0.703 | |
6 | GAD | 0.474 | GAD | 0.531 | SCPL | 0.743 | |
7 | BLH1 | 0.526 | BLH1 | 0.55 | SBT3.17 | 0.855 | |
8 | HMGB2 | 0.7 | SBT3.17 | 0.802 | GEPI48 | 0.945 | |
9 | ALEU | 1.004 | GEPI48 | 1.008 | ALEU | 1.996 |
Category | Rank | Leaves under Different Temperature Stresses | Leaves of Plants at Different Growth Stages | Different Organs | ||||||
---|---|---|---|---|---|---|---|---|---|---|
Gene | SD * | CV * | Gene | SD | CV | Gene | SD | CV | ||
Candidate traditional house-keeping genes | 1 | eIF5A | 0.32 | 1.31 | TUB | 0.33 | 1.29 | Actin7 | 0.52 | 2.07 |
2 | UBC2 | 0.36 | 1.26 | GAPDH | 0.33 | 1.4 | eIF5A | 0.52 | 2.22 | |
3 | GAPDH | 0.36 | 1.37 | eIF5A | 0.34 | 1.53 | UBQ | 0.61 | 2.31 | |
4 | TUA | 0.45 | 1.61 | TUA | 0.43 | 1.68 | TUA | 0.67 | 2.59 | |
5 | UBQ | 0.61 | 2.65 | FtsH2 | 0.49 | 2.02 | TUB | 0.82 | 3.11 | |
6 | TUB | 0.67 | 2.35 | ClpA | 0.5 | 2.48 | GAPDH | 0.85 | 3.32 | |
7 | CYP72A | 0.75 | 2.77 | CYP72A | 0.53 | 2.21 | eIF4A | 0.91 | 3.49 | |
8 | ClpA | 0.8 | 3.7 | UBQ | 0.53 | 2.5 | FtsH2 | 1 | 3.56 | |
9 | RAP2 | 0.9 | 3.16 | eIF4A | 0.57 | 2.3 | ClpA | 1.09 | 4.63 | |
10 | eIF4A | 0.9 | 3.41 | Actin7 | 0.7 | 2.84 | RAP2 | 1.25 | 4.4 | |
11 | FtsH2 | 1.19 | 4.53 | RAP2 | 0.8 | 2.97 | UBC2 | 1.29 | 4.6 | |
12 | Actin7 | 1.24 | 4.89 | UBC2 | 0.81 | 3.05 | CYP72A | 1.44 | 5.3 | |
Candidate new RGs | 1 | HMGB2 | 0.65 | 2.64 | HMGB2 | 0.38 | 1.63 | SCPL | 0.5 | 1.9 |
2 | SBT3.17 | 0.74 | 2.89 | RTNLB1 | 0.42 | 1.77 | SBT3.17 | 0.57 | 2.23 | |
3 | SCPL | 0.79 | 3.02 | ALEU | 0.42 | 1.81 | HMGB2 | 0.71 | 2.9 | |
4 | GAD | 0.84 | 3.27 | GAD | 0.46 | 1.97 | GAD | 0.77 | 2.89 | |
5 | RTNLB1 | 0.91 | 3.55 | UNC | 0.5 | 1.97 | RTNLB1 | 0.85 | 3.23 | |
6 | BLH1 | 0.92 | 3.69 | BLH1 | 0.56 | 2.39 | BLH1 | 0.88 | 3.31 | |
7 | GEPI48 | 1.06 | 4.16 | SCPL | 0.6 | 2.35 | UNC | 0.9 | 3.27 | |
8 | UNC | 1.11 | 4.11 | SBT3.17 | 0.69 | 2.78 | GEPI48 | 0.9 | 3.42 | |
9 | ALEU | 1.15 | 4.58 | GEPI48 | 0.73 | 3.01 | ALEU | 1.47 | 5.56 |
Category | Rank | Leaves under Different Temperature Stresses | Leaves of Plants at Different Growth Stages | Different Organs | |||
---|---|---|---|---|---|---|---|
Gene | Stability Value | Gene | Stability Value | Gene | Stability Value | ||
Candidate traditional house-keeping genes | 1 | eIF5A | 1.41 | TUB | 1.41 | eIF5A | 1.68 |
2 | TUA | 2 | eIF5A | 1.97 | Actin7 | 2.63 | |
3 | UBC2 | 2.38 | TUA | 2.99 | TUA | 3.13 | |
4 | GAPDH | 3.87 | GAPDH | 3.35 | GAPDH | 3.31 | |
5 | CYP72A | 4.21 | FtsH2 | 4.95 | ClpA | 5.05 | |
6 | TUB | 6.48 | ClpA | 5.63 | TUB | 5.21 | |
7 | ClpA | 7.44 | eIF4A | 5.8 | FtsH2 | 6.16 | |
8 | eIF4A | 7.61 | CYP72A | 7.97 | UBQ | 6.51 | |
9 | UBQ | 8.61 | Actin7 | 8.97 | eIF4A | 7.94 | |
10 | Actin7 | 9.67 | UBQ | 9.69 | RAP2 | 7.95 | |
11 | FtsH2 | 10.74 | RAP2 | 10.74 | CYP72A | 11.24 | |
12 | RAP2 | 11.17 | UBC2 | 12 | UBC2 | 11.74 | |
Candidate new RGs | 1 | SCPL | 1.86 | ALEU | 1.86 | HMGB2 | 1.73 |
2 | RTNLB1 | 2.78 | RTNLB1 | 2 | SCPL | 2.21 | |
3 | GEPI48 | 3.03 | HMGB2 | 2.34 | SBT3.17 | 3.15 | |
4 | SBT3.17 | 3.31 | GAD | 3.31 | RTNLB1 | 3.5 | |
5 | UNC | 3.76 | UNC | 4.16 | UNC | 3.6 | |
6 | HMGB2 | 4.76 | SCPL | 5.45 | GAD | 4.68 | |
7 | GAD | 5.42 | BLH1 | 6.48 | BLH1 | 5.96 | |
8 | BLH1 | 6.74 | SBT3.17 | 8 | GEPI48 | 8 | |
9 | ALEU | 9 | GEPI48 | 9 | ALEU | 9 |
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Guo, W.; Yang, Y.; Ma, B.; Wang, W.; Hu, Z.; Leng, P. Selection and Validation of Reference Genes for Gene Expression Studies in Euonymus japonicus Based on RNA Sequencing. Genes 2024, 15, 131. https://doi.org/10.3390/genes15010131
Guo W, Yang Y, Ma B, Wang W, Hu Z, Leng P. Selection and Validation of Reference Genes for Gene Expression Studies in Euonymus japonicus Based on RNA Sequencing. Genes. 2024; 15(1):131. https://doi.org/10.3390/genes15010131
Chicago/Turabian StyleGuo, Wei, Yihui Yang, Bo Ma, Wenbo Wang, Zenghui Hu, and Pingsheng Leng. 2024. "Selection and Validation of Reference Genes for Gene Expression Studies in Euonymus japonicus Based on RNA Sequencing" Genes 15, no. 1: 131. https://doi.org/10.3390/genes15010131
APA StyleGuo, W., Yang, Y., Ma, B., Wang, W., Hu, Z., & Leng, P. (2024). Selection and Validation of Reference Genes for Gene Expression Studies in Euonymus japonicus Based on RNA Sequencing. Genes, 15(1), 131. https://doi.org/10.3390/genes15010131