Screening Potential Reference Genes in Tuta absoluta with Real-Time Quantitative PCR Analysis under Different Experimental Conditions
Abstract
:1. Introduction
2. Materials and Methods
2.1. Insects
2.2. Treatments
2.2.1. Developmental Stages
2.2.2. Adult Tissues
2.2.3. Insecticides
2.2.4. Temperatures
2.2.5. Plant Volatile Organic Compounds (VOCs)
2.3. Total RNA Extraction and cDNA Synthesis
2.4. Candidate Reference Gene Selection and Specific Primer Design
2.5. Standard Curve Construction and RT-qPCR
2.6. Statistical Analysis
3. Results
3.1. Acquisition of Candidate Reference Gene Sequences
3.2. Specificity and Amplification Efficiency of Specific RT-qPCR Primers
3.3. Expression Levels of the Candidate Reference Genes
3.4. Expression Stability and Ranking of the Candidate Reference Genes
3.4.1. Developmental Stages
3.4.2. Adult Tissues
3.4.3. Insecticide-Induced Stress
3.4.4. Temperature-Induced Stress
3.4.5. Plant VOC-Induced Stress
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
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Gene Name | Primer | Primer Sequences (5′-3′) | Tm (°C) | Amplicon Length (bp) |
---|---|---|---|---|
TUB | TUB-F TUB-R | GAGTGCATCTCAGTCCACGTT GAAGGAGTGGAAGATGAGGAAT | 55 | 417 |
β-actin | β-actin-F β-actin-R | TCCTTGGAGATCCACATCTG CCCATCTACGAAGGTTACG | 53 | 590 |
EF1-α | EF1-α-F EF1-α-R | GCCCTTCTCGCCTTCACCCTT GGGCACCGTTCCAATACCACC | 62 | 381 |
RPL27 | RPL27-F RPL27-R | GTAAGATTATGAAACCTGGGAA ACCTAAGCTTTTGGAAGAACC | 51 | 395 |
RPS13 | RPS13-F RPS13-R | CCCTACCTGGTTGAAACTGAC TGAAGCGGTGCTAGACTCATA | 51 | 376 |
TBP | TBP-F TBP-R | TCCTCACCACTTGTCGGCT GTTGTCCGTGGCTCTCTGAT | 58 | 374 |
RPS15 | RPS15-F RPS15-R | CGAGAAATACCCCAGAAATGT TTCTTCGCTTGTTGTTTGATT | 51 | 429 |
Primer | Primer Sequences (5′-3′, F/R) | Amplicon Length (bp) | Tm (°C) | Efficiency (%) | R2 |
---|---|---|---|---|---|
RPL27 | CGTTACTCGGTAGACTTCA GTTCTTTCCACTCTTGTATCG | 126 | 60 | 92.7 | 0.992 |
RPS13 | AGTTGCCCAAGTCAGATT TTCCAAGTGCTTCCTCAT | 133 | 60 | 102.0 | 0.999 |
RPS15 | ATACGAAGTGAACAGTTAGC ACGGTCCATTCTTCCAAT | 92 | 60 | 103.8 | 0.990 |
EF1-α | AGTCTCCTCATACATCAAGAAG CCTCCTTACGCTCAACAG | 143 | 60 | 100.7 | 0.998 |
TBP | GTCTACTCCACACCTCAA CTGCCACTCGTTATCATTG | 77 | 60 | 110.2 | 0.993 |
TUB | GGAGTCCAGATCGGTAAC GCTGAAGAAGGTGTTGAAC | 126 | 60 | 95.9 | 0.998 |
β-actin | CAGGTCCTTACGGATGTC CTCTTCCAGCCTTCCTTC | 99 | 60 | 104.4 | 0.996 |
Gene Name | Total Samples | Developmental Stage | Tissue | Insecticide | Temperature | Plant VOC | |||
---|---|---|---|---|---|---|---|---|---|
Treatment | Control | Treatment | Control | Treatment | Control | ||||
TBP | 25.84 ± 1.28 b | 25.14 ± 1.15 b | 25.45 ± 0.98 b | 26.92 ± 0.51 a | 27.64 ± 0.52 a | 27.80 ± 0.65 a | 27.84 ± 0.64 a | 25.61 ± 0.39 b | 25.54 ± 0.35 b |
TUB | 21.14 ± 2.22 d | 19.31 ± 1.27 d,e | 22.81 ± 1.16 c | 20.41 ± 2.29 b,c | 21.34 ± 0.44 d | 21.78 ± 0.60 d | 21.63 ± 0.74 d | 24.09 ± 0.51 c,d | 23.88 ± 0.47 d |
EF1-α | 20.19 ± 1.50 d,e | 19.80 ± 1.04 d | 20.30 ± 1.47 d | 19.22 ± 1.04 c | 20.12 ± 0.83 e | 19.74 ± 0.89 e | 19.92 ± 0.85 e | 22.74 ± 0.69 e | 22.35 ± 0.37 e |
β-actin | 19.36 ± 3.68 e | 18.11 ± 3.69 e | 22.42 ± 1.41 c | 16.33 ± 1.70 d | 16.50 ± 0.15 f | 16.71 ± 0.25 f | 16.58 ± 0.21 f | 23.83 ± 0.63 d | 23.69 ± 0.59 d |
RPS13 | 22.61 ± 1.22 c | 22.30 ± 1.00 c | 22.83 ± 0.95 c | 22.16 ± 0.49 b | 21.28 ± 0.46 d | 21.44 ± 0.55 d | 21.46 ± 0.58 d | 24.72 ± 0.50 c | 24.51 ± 0.41 c |
RPS15 | 26.08 ± 1.62 b | 25.35 ± 1.31 b | 26.94 ± 1.14 b | 27.39 ± 1.91 a | 26.57 ± 0.53 b | 26.93 ± 0.70 b | 26.69 ± 0.53 b | 24.44 ± 0.63 c,d | 24.08 ± 0.41 c,d |
RPL27 | 27.47 ± 1.84 a | 27.49 ± 1.15 a | 28.88 ± 2.34 a | 27.63 ± 0.74 a | 24.17 ± 0.57 c | 24.37 ± 0.61 c | 24.38 ± 0.69 c | 27.45 ± 0.76 a | 26.99 ± 0.42 a |
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Yang, A.-P.; Wang, Y.-S.; Huang, C.; Lv, Z.-C.; Liu, W.-X.; Bi, S.-Y.; Wan, F.-H.; Wu, Q.; Zhang, G.-F. Screening Potential Reference Genes in Tuta absoluta with Real-Time Quantitative PCR Analysis under Different Experimental Conditions. Genes 2021, 12, 1253. https://doi.org/10.3390/genes12081253
Yang A-P, Wang Y-S, Huang C, Lv Z-C, Liu W-X, Bi S-Y, Wan F-H, Wu Q, Zhang G-F. Screening Potential Reference Genes in Tuta absoluta with Real-Time Quantitative PCR Analysis under Different Experimental Conditions. Genes. 2021; 12(8):1253. https://doi.org/10.3390/genes12081253
Chicago/Turabian StyleYang, An-Pei, Yu-Sheng Wang, Cong Huang, Zhi-Chuang Lv, Wan-Xue Liu, Si-Yan Bi, Fang-Hao Wan, Qiang Wu, and Gui-Fen Zhang. 2021. "Screening Potential Reference Genes in Tuta absoluta with Real-Time Quantitative PCR Analysis under Different Experimental Conditions" Genes 12, no. 8: 1253. https://doi.org/10.3390/genes12081253
APA StyleYang, A.-P., Wang, Y.-S., Huang, C., Lv, Z.-C., Liu, W.-X., Bi, S.-Y., Wan, F.-H., Wu, Q., & Zhang, G.-F. (2021). Screening Potential Reference Genes in Tuta absoluta with Real-Time Quantitative PCR Analysis under Different Experimental Conditions. Genes, 12(8), 1253. https://doi.org/10.3390/genes12081253