Expression Analysis of XTH in Stem Swelling of Stem Mustard and Selection of Reference Genes
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plant RNA Extraction and cDNA Synthesis
2.2. Selection of Reference Genes, Primer Design, and Cloning
2.3. Real-Time Quantitative PCR Amplification (qPCR)
2.4. Data Analysis and Evaluation of Stability of Reference Genes
2.5. Identification of XTH Family Genes in B. juncea
2.6. Expression Analysis and Validation of XTH Using qPCR
3. Results
3.1. Specificity and Efficiency in Amplification of Candidate Reference Genes
3.2. Evaluation of Expression Stability of Reference Genes
3.2.1. Cq Value of Candidate Reference Genes
3.2.2. geNorm Software Analysis
3.2.3. NormFinder Software Analysis
3.2.4. BestKeeper Software Analysis
3.2.5. RefFinder Software Analysis
3.3. Determination of the Optimal Number of Reference Genes
3.4. Validation of Reference Genes Using qPCR
3.5. Identification and Phylogenetic Analysis of XTH Gene Family in B. juncea
3.6. Gene Expression Analysis of Mustard XTH Family
4. Discussion
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Gene Symbol | Gene Name | Primer Sequence (5’-3’) | Amplicon Size (bp) | Correlation Coefficient |
---|---|---|---|---|
EF-1α | Elongation factor -1α gene | CGTCTGCTTAGTGAACCTGCTC/ GAAGGAGCGAATGTCACAACCA | 112 | 0.996 |
HIS | Histone | AGGGAAAGCCGCTCCACTTC/ TCGTAACCCTCTTGGCGTGAAT | 152 | 0.996 |
IF-4α | Eukaryotic initiation factor 4 alpha | CAAGCCGAGCCTGCGATCAT/ TCGTGTTCTGGTCCATGTCTCC | 121 | 0.996 |
PP2A | Protein phosphatase 2A | AAGCCGAGCCTTCCATCATAGC/ ACCACCACCACCACTCATTGC | 197 | 0.992 |
TUB | Tubulin gene | GCGTCTTGTCCGTGAGATTGC/ GCCGAGATGAGGTGGTTAAGGT | 109 | 0.994 |
UBC | Ubiquitin C gene | GCCATCACTCAGAGCGTCATCT/ AAGGAGACTGTGTAGGACCAAGAA | 100 | 0.999 |
UBQ | Polyubiquitin 10 gene | TGCGTCTACCACTTCAGGATGT/ TCTGCTGGTCTGGAGGAATGC | 145 | 0.992 |
ACTIN | Actin gene | ATCGTCTGTGACAACGGTAC/ ATGGAGGGTGATGAGATTCAGC | 267 | 0.992 |
Group | Rank | geNorm | NormFinder | BestKeeper | ||||
---|---|---|---|---|---|---|---|---|
Gene | Stability | Gene Name | Stability Value | Gene | SD [±Cq] | CV [%Cq] | ||
Total | 1 | UBC | 0.49 | UBC | 0.31 | TUB | 0.67 | 2.73 |
2 | UBQ | 0.49 | TUB | 0.39 | UBC | 0.77 | 3.02 | |
3 | TUB | 0.71 | UBQ | 0.48 | UBQ | 0.8 | 3.74 | |
4 | HIS | 0.92 | HIS | 0.59 | ACTIN | 0.86 | 2.91 | |
5 | EF-1α | 1.03 | IF-4α | 0.68 | HIS | 0.87 | 3.51 | |
6 | IF-4α | 1.06 | EF-1α | 0.7 | EF-1α | 0.9 | 4.01 | |
7 | PP2A | 1.22 | PP2A | 0.97 | IF-4α | 0.92 | 3.83 | |
8 | ACTIN | 1.36 | ACTIN | 1.06 | PP2A | 1 | 3.53 | |
S1 | 1 | UBC | 0.33 | UBC | 0.11 | EF-1α | 0.28 | 1.26 |
2 | TUB | 0.33 | IF-4α | 0.17 | IF-4α | 0.39 | 1.61 | |
3 | UBQ | 0.45 | TUB | 0.26 | HIS | 0.53 | 2.13 | |
4 | IF-4α | 0.52 | UBQ | 0.45 | PP2A | 0.66 | 2.26 | |
5 | PP2A | 0.61 | EF-1α | 0.45 | UBC | 0.71 | 2.67 | |
6 | EF-1α | 0.69 | PP2A | 0.45 | TUB | 0.81 | 3.29 | |
7 | HIS | 0.73 | HIS | 0.56 | UBQ | 0.9 | 4.01 | |
8 | ACTIN | 1.15 | ACTIN | 1.65 | ACTIN | 1.83 | 6.05 | |
S2 | 1 | UBC | 0.26 | IF-4α | 0.19 | TUB | 0.25 | 1.02 |
2 | HIS | 0.26 | UBC | 0.23 | IF-4α | 0.62 | 2.78 | |
3 | EF-1α | 0.37 | UBQ | 0.25 | ACTIN | 0.7 | 2.39 | |
4 | UBQ | 0.44 | EF-1α | 0.35 | UBQ | 0.86 | 4.09 | |
5 | IF-4α | 0.5 | TUB | 0.39 | UBC | 0.93 | 3.75 | |
6 | TUB | 0.58 | HIS | 0.42 | EF-1α | 1.05 | 4.9 | |
7 | PP2A | 0.71 | PP2A | 0.62 | HIS | 1.05 | 4.37 | |
8 | ACTIN | 0.88 | ACTIN | 0.93 | PP2A | 1.14 | 3.89 | |
S3 | 1 | UBC | 0.4 | EF-1α | 0.11 | EF-1α | 0.23 | 0.96 |
2 | TUB | 0.4 | UBC | 0.27 | UBC | 0.36 | 1.4 | |
3 | UBQ | 0.43 | PP2A | 0.33 | PP2A | 0.5 | 1.85 | |
4 | EF-1α | 0.48 | IF-4α | 0.38 | UBQ | 0.51 | 2.39 | |
5 | IF-4α | 0.61 | UBQ | 0.46 | TUB | 0.6 | 2.37 | |
6 | PP2A | 0.68 | TUB | 0.55 | IF-4α | 0.62 | 2.54 | |
7 | HIS | 0.82 | HIS | 0.68 | HIS | 0.73 | 2.84 | |
8 | ACTIN | 0.9 | ACTIN | 0.68 | ACTIN | 0.84 | 2.84 | |
S4 | 1 | UBC | 0.25 | UBC | 0.08 | EF-1α | 0.38 | 1.68 |
2 | TUB | 0.25 | TUB | 0.23 | PP2A | 0.57 | 2.09 | |
3 | IF-4α | 0.32 | EF-1α | 0.27 | IF-4α | 0.58 | 2.35 | |
4 | UBQ | 0.4 | IF-4α | 0.32 | UBC | 0.6 | 2.39 | |
5 | EF-1α | 0.48 | PP2A | 0.37 | TUB | 0.68 | 2.81 | |
6 | PP2A | 0.58 | UBQ | 0.37 | UBQ | 0.83 | 3.99 | |
7 | HIS | 0.65 | HIS | 0.55 | HIS | 0.94 | 3.83 | |
8 | ACTIN | 0.86 | ACTIN | 0.96 | ACTIN | 1.05 | 3.61 |
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Li, M.; Xie, F.; He, Q.; Li, J.; Liu, J.; Sun, B.; Luo, Y.; Zhang, Y.; Chen, Q.; Zhang, F.; et al. Expression Analysis of XTH in Stem Swelling of Stem Mustard and Selection of Reference Genes. Genes 2020, 11, 113. https://doi.org/10.3390/genes11010113
Li M, Xie F, He Q, Li J, Liu J, Sun B, Luo Y, Zhang Y, Chen Q, Zhang F, et al. Expression Analysis of XTH in Stem Swelling of Stem Mustard and Selection of Reference Genes. Genes. 2020; 11(1):113. https://doi.org/10.3390/genes11010113
Chicago/Turabian StyleLi, Mengyao, Fangjie Xie, Qi He, Jie Li, Jiali Liu, Bo Sun, Ya Luo, Yong Zhang, Qing Chen, Fen Zhang, and et al. 2020. "Expression Analysis of XTH in Stem Swelling of Stem Mustard and Selection of Reference Genes" Genes 11, no. 1: 113. https://doi.org/10.3390/genes11010113
APA StyleLi, M., Xie, F., He, Q., Li, J., Liu, J., Sun, B., Luo, Y., Zhang, Y., Chen, Q., Zhang, F., Gong, R., Wang, Y., Wang, X., & Tang, H. (2020). Expression Analysis of XTH in Stem Swelling of Stem Mustard and Selection of Reference Genes. Genes, 11(1), 113. https://doi.org/10.3390/genes11010113