Reference Genes Selection and Validation for Cinnamomum burmanni by Real-Time Quantitative Polymerase Chain Reaction
Abstract
:1. Introduction
2. Results
2.1. Primer Specificity and Amplification Efficiency of Candidate Reference Genes
2.2. Expression Analysis of Candidate Reference Genes of C. burmannii
2.3. Gene Expression Stability Analysis
2.4. Reference Gene Validation
3. Discussion
4. Materials and Methods
4.1. Plant Materials
4.2. RNA Extraction and cDNA Synthesis
4.3. Candidate Reference Genes Selection and Primer Design
4.4. q-PCR Amplification
4.5. Data Analysis and Validation of Selected Reference Genes
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Gene-ID | Gene Abbreviation | Tentative Annotation | Primer Sequence of Forward | Primer Sequence of Reward | Amplicon Length (bp) | Tm (°C) | E | R2 |
---|---|---|---|---|---|---|---|---|
Cbur01G028330 | ACT7 | actin7 | CAACCCAAAAGCCAACAGG | TCACCCGAGTCCAGAACAATAC | 141 | 58.7/59.1 | 98.76% | 0.9968 |
Cbur02G019900 | Cpn60β | chaperonin 60 subunit beta 2 | CAACAAGGATGGGCTGGCTA | TTGGCCACAGTCACTCCATC | 156 | 60/60 | 98.05% | 0.9979 |
Cbur01G001170 | EF1α | elongation factor 1-alpha | GGTACAAGGGCCCAACTCTC | CTGGAGAGCTTCATGGTGCA | 236 | 60/60 | 89.99% | 0.9983 |
Cbur05G032970 | eIF-5A | eukaryotic translation initiation factor 5A | CCAAGTGTCACTTTGTGGCG | AGTGGGGAGCCTCAGATCAT | 191 | 60/60 | 86.05% | 0.9993 |
Cbur10G024220 | GAPDH | glyceraldehyde-3-phosphate dehydrogenase | AAGGGTGGTGCCAAGAAAGT | GTTGCAGTGATGGAGTGGACAG | 215 | 58.6/60.2 | 92.81% | 0.9917 |
Cbur06G016220 | GIIα | glucan 1,3-alpha-glucosidase | CCTTATCGCCTTTTCAACCTT | AGCGTATCAATCCGCCCTC | 221 | 58.3/59.9 | 90.63% | 0.9983 |
Cbur08G011150 | HIS | histone superfamily protein H3 | GGAGGGAAGGCTCCTAGGAA | CAACTGTTCCAGGGCGGTAT | 106 | 60/60 | 96.01% | 0.9985 |
Cbur10G000690 | RA | rubisco activase | ACAGACCGACAAGGACAAATGG | CGGAGACCCGTGCTCAAGTAT | 168 | 61.3/61.6 | 79.95% | 0.9926 |
Cbur10G003920 | RPL27 | ribosomal protein L27 | GCCGTCATCGTACGATCCTT | TGCCGTCTTCTTTGCAGAGT | 123 | 60.0/59.9 | 98.39% | 0.9969 |
Cbur07G013210 | RPS15 | ribosomal protein S15 | GCAGCCGAAGAGGAGAACA | GGCTTCCGCTTCAAACCAC | 144 | 58.4//60.4 | 92.04% | 0.9972 |
Cbur04G009020 | TATA | TATA-box-binding protein | CCGTAATGCAGAGTATAACCCC | TTTGACATCACAAGAGCCCAC | 146 | 60.1/59.5 | 82.13% | 0.9989 |
Cbur08G006150 | TUB | tubulin β chain | TGGGAATAACTGGGCTAAGGG | AAGCATCATCCGATCAGGGTA | 205 | 60.9/59.5 | 95.11% | 0.9964 |
Cbur02G028660 | APT | adenine phosphoribosy ltransferase 1 | TGCTTGATCCCGAGGCATTT | ACTTCGAACCAAGGGCCAAA | 141 | 60.1/60 | 89.03% | 0.9993 |
Total | Cold-treated | Nacl-treated | PEG-treated | Tissues | Leaves at Different Developmental Stages | Different Borneol Clones | |||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Gene | mSD | Gene | mSD | Gene | mSD | Gene | mSD | Gene | mSD | Gene | mSD | Gene | mSD |
ACT7 | 1.21 | ACT7 | 0.51 | ACT7 | 0.87 | ACT7 | 0.62 | ACT7 | 1.30 | ACT7 | 0.94 | ACT7 | 1.56 |
APT | 1.29 | APT | 0.49 | APT | 1.14 | APT | 0.64 | APT | 1.93 | APT | 1.14 | APT | 1.22 |
Cpn60β | 1.61 | Cpn60β | 0.63 | Cpn60β | 1.37 | Cpn60β | 1.09 | Cpn60β | 1.81 | Cpn60β | 1.43 | Cpn60β | 2.72 |
EF1α | 1.22 | EF1α | 0.63 | EF1α | 1.41 | EF1α | 0.49 | EF1α | 1.29 | EF1α | 1.10 | EF1α | 1.10 |
eIF-5A | 1.10 | eIF-5A | 0.58 | eIF-5A | 0.95 | eIF-5A | 0.55 | eIF-5A | 1.52 | eIF-5A | 1.32 | eIF-5A | 0.94 |
GAPDH | 1.37 | GAPDH | 0.49 | GAPDH | 1.24 | GAPDH | 0.68 | GAPDH | 1.67 | GAPDH | 1.67 | GAPDH | 1.69 |
Gllα | 1.13 | Gllα | 0.62 | Gllα | 0.90 | Gllα | 0.75 | Gllα | 1.91 | Gllα | 0.94 | Gllα | 0.93 |
HIS | 1.31 | HIS | 0.66 | HIS | 1.14 | HIS | 0.81 | HIS | 1.73 | HIS | 1.14 | HIS | 1.01 |
RA | 3.01 | RA | 0.71 | RA | 1.61 | RA | 0.98 | RA | 6.61 | RA | 3.17 | RA | 1.26 |
RPL27 | 1.05 | RPL27 | 0.43 | RP L27 | 0.85 | RP L27 | 0.48 | RP L27 | 1.32 | RPL27 | 0.93 | RPL27 | 1.30 |
RPS15 | 0.98 | RPS15 | 0.46 | RPS15 | 0.83 | RPS15 | 0.47 | RPS15 | 1.36 | RPS15 | 0.93 | RPS15 | 0.90 |
TATA | 1.04 | TATA | 0.57 | TATA | 0.92 | TATA | 0.49 | TATA | 1.31 | TATA | 0.95 | TATA | 1.11 |
TUB | 1.34 | TUB | 0.89 | TUB | 0.97 | TUB | 0.51 | TUB | 2.03 | TUB | 2.16 | TUB | 1.05 |
Total | Cold-treated | Nacl-treated | PEG-treated | Tissues | Leaves at Different Developmental Stages | Different Borneol Clones | |||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Gene | SV | Gene | SV | Gene | SV | Gene | SV | Gene | SV | Gene | SV | Gene | SV |
ACT7 | 0.811 | ACT7 | 0.285 | ACT7 | 0.438 | ACT7 | 0.401 | ACT7 | 0.058 | ACT7 | 0.063 | ACT7 | 1.482 |
APT | 0.877 | APT | 0.247 | APT | 0.934 | APT | 0.448 | APT | 1.380 | APT | 0.547 | APT | 1.007 |
Cpn60β | 1.314 | Cpn60β | 0.506 | Cpn60β | 1.114 | Cpn60β | 1.005 | Cpn60β | 1.445 | Cpn60β | 1.034 | Cpn60β | 2.660 |
EF1α | 0.745 | EF1α | 0.476 | EF1α | 1.311 | EF1α | 0.102 | EF1α | 0.058 | EF1α | 0.439 | EF1α | 0.499 |
eIF-5A | 0.436 | eIF-5A | 0.427 | eIF-5A | 0.559 | eIF-5A | 0.308 | eIF-5A | 0.208 | eIF-5A | 0.834 | eIF-5A | 0.128 |
GAPDH | 0.995 | GAPDH | 0.255 | GAPDH | 1.082 | GAPDH | 0.521 | GAPDH | 1.205 | GAPDH | 1.448 | GAPDH | 1.440 |
Gllα | 0.564 | Gllα | 0.473 | Gllα | 0.429 | Gllα | 0.603 | Gllα | 1.390 | Gllα | 0.131 | Gllα | 0.138 |
HIS | 0.834 | HIS | 0.545 | HIS | 0.899 | HIS | 0.673 | HIS | 0.948 | HIS | 0.617 | HIS | 0.542 |
RA | 2.918 | RA | 0.593 | RA | 1.499 | RA | 0.887 | RA | 6.568 | RA | 3.132 | RA | 0.760 |
RPL27 | 0.467 | RPL27 | 0.115 | RPL27 | 0.328 | RPL27 | 0.114 | RPL27 | 0.126 | RPL27 | 0.131 | RPL27 | 1.134 |
RPS15 | 0.210 | RPS15 | 0.171 | RPS15 | 0.340 | RPS15 | 0.082 | RPS15 | 0.099 | RPS15 | 0.063 | RPS15 | 0.153 |
TATA | 0.344 | TATA | 0.378 | TATA | 0.570 | TATA | 0.107 | TATA | 0.099 | TATA | 0.119 | TATA | 0.700 |
TUB | 0.969 | TUB | 0.803 | TUB | 0.601 | TUB | 0.194 | TUB | 1.644 | TUB | 2.086 | TUB | 0.426 |
Total | Cold-treated | Nacl-treated | PEG-treated | Tissues | Leaves at Different Developmental Stages | Different Borneol Clones | |||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Gene | SD [±CP] | Gene | SD [±CP] | Gene | SD [±CP] | Gene | SD [±CP] | Gene | SD [±CP] | Gene | SD [±CP] | Gene | SD [±CP] |
ACT7 | 0.56 | ACT7 | 0.58 | ACT7 | 0.52 | ACT7 | 0.58 | ACT7 | 0.19 | ACT7 | 0.79 | ACT7 | 0.53 |
APT | 0.73 | APT | 0.27 | APT | 1.13 | APT | 0.54 | APT | 0.85 | APT | 0.60 | APT | 0.32 |
Cpn60β | 1.00 | Cpn60β | 0.56 | Cpn60β | 0.83 | Cpn60β | 0.38 | Cpn60β | 0.61 | Cpn60β | 1.43 | Cpn60β | 2.37 |
EF1α | 0.78 | EF1α | 0.36 | EF1α | 1.28 | EF1α | 0.38 | EF1α | 0.26 | EF1α | 1.03 | EF1α | 0.96 |
eIF-5A | 0.64 | eIF-5A | 0.42 | eIF-5A | 0.66 | eIF-5A | 0.40 | eIF-5A | 0.83 | eIF-5A | 0.46 | eIF-5A | 0.63 |
GAPDH | 0.96 | GAPDH | 0.37 | GAPDH | 1.21 | GAPDH | 0.57 | GAPDH | 0.32 | GAPDH | 1.80 | GAPDH | 1.45 |
Gllα | 0.76 | Gllα | 0.67 | Gllα | 0.75 | Gllα | 0.59 | Gllα | 1.04 | Gllα | 1.09 | Gllα | 0.62 |
HIS | 1.09 | HIS | 0.74 | HIS | 1.21 | HIS | 0.71 | HIS | 0.79 | HIS | 1.38 | HIS | 0.10 |
RA | 1.87 | RA | 0.53 | RA | 0.68 | RA | 0.80 | RA | 4.74 | RA | 1.20 | RA | 0.89 |
RPL27 | 0.47 | RPL27 | 0.35 | RPL27 | 0.44 | RPL27 | 0.35 | RPL27 | 0.53 | RPL27 | 1.01 | RPL27 | 0.43 |
RPS15 | 0.53 | RPS15 | 0.55 | RPS15 | 0.45 | RPS15 | 0.36 | RPS15 | 0.56 | RPS15 | 0.87 | RPS15 | 0.41 |
TATA | 0.57 | TATA | 0.48 | TATA | 0.38 | TATA | 0.43 | TATA | 0.52 | TATA | 0.79 | TATA | 0.26 |
TUB | 0.83 | TUB | 0.55 | TUB | 0.65 | TUB | 0.37 | TUB | 1.05 | TUB | 2.38 | TUB | 0.80 |
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Shi, L.; Cai, Y.; Yao, J.; Zhang, Q.; He, B.; Lin, S. Reference Genes Selection and Validation for Cinnamomum burmanni by Real-Time Quantitative Polymerase Chain Reaction. Int. J. Mol. Sci. 2024, 25, 3500. https://doi.org/10.3390/ijms25063500
Shi L, Cai Y, Yao J, Zhang Q, He B, Lin S. Reference Genes Selection and Validation for Cinnamomum burmanni by Real-Time Quantitative Polymerase Chain Reaction. International Journal of Molecular Sciences. 2024; 25(6):3500. https://doi.org/10.3390/ijms25063500
Chicago/Turabian StyleShi, Lingling, Yanling Cai, Jun Yao, Qian Zhang, Boxiang He, and Shanzhi Lin. 2024. "Reference Genes Selection and Validation for Cinnamomum burmanni by Real-Time Quantitative Polymerase Chain Reaction" International Journal of Molecular Sciences 25, no. 6: 3500. https://doi.org/10.3390/ijms25063500