Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (10): 80-89.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1616
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XU Yuan-yuan1,2,3(), ZHAO Guo-chun1,2,3, HAO Ying-ying1,2,3, WENG Xue-huang4, CHEN Zhong1,2,3,5(), JIA Li-ming1,2,3()
Received:
2021-12-30
Online:
2022-10-26
Published:
2022-11-11
Contact:
CHEN Zhong,JIA Li-ming
E-mail:yuanyuanxu_2016@163.com;zhongchen@bjfu.edu.cn;jlm@bjfu.edu.cn
XU Yuan-yuan, ZHAO Guo-chun, HAO Ying-ying, WENG Xue-huang, CHEN Zhong, JIA Li-ming. Reference Genes Selection and Validation for RT-qPCR in Sapindus mukorossi[J]. Biotechnology Bulletin, 2022, 38(10): 80-89.
Fig. 1 Sample materials of S. mukorossi R:root;ST:stem;L:leaf;B:bud;MF:male flower;FF:female flower;S1:pericarp at 15 d after flower pollination;S2:pericarp at 45 d after flower pollination;S3:pericarp at 75 d after flower pollination;S4:pericarp at 90 d after flower pollination;S5:pericarp at 105 d after flower pollination;S6:pericarp at 120 d after flower pollination;S7:pericarp at 135 d after flower pollination;S8:pericarp at 150 d after flower pollination
内参基因 Reference genes | 基因ID Gene ID | 产物长度 Length of product /bp | 正向引物序列 Forward primer sequences(5'- 3') | 反向引物序列 Reverse primer sequences(5'- 3') |
---|---|---|---|---|
Sm18S | Samuk09G0136600 | 221 | AGATTGAAGGCGTTCTTTGGAT | TCAGTGTTTATGGACGGTGGAC |
SmACT | Samuk13G0061200 | 129 | AGAAAGTTGGCCTCGCTGAA | CAGGAACCAGACCACCTGTC |
SmEF-1α | Samuk14G0055700 | 101 | TCCAAGGCCAGGTACGATGA | AAACCAGAGATGGGGACGAAG |
SmRPL1 | Samuk07G0011500 | 206 | CTGACCACCCCACCACTCTC | TCCTCCTCATCTGCCACCTC |
SmRPS26 | Samuk08G0043900 | 230 | GCGAAGAAATGGAGGAAGGA | AGTGGATGGCACACGAAACA |
SmTBCC | Samuk01G0035500 | 130 | AGCCTGACACCCTTTCTCTACC | CCAACCTCTTCTGCAGCCTT |
SmUBC12 | Samuk11G0037500 | 102 | TTGTCTGGACCAACCAAAGGA | TGCTTCTTGCCAGGTGTTTTC |
SmUBP | Samuk09G0089700 | 180 | TGCACAGTTGTTGCTCATGC | CTGCTTCATTTTCCCCGTGC |
Table 1 Primers of candidate reference genes
内参基因 Reference genes | 基因ID Gene ID | 产物长度 Length of product /bp | 正向引物序列 Forward primer sequences(5'- 3') | 反向引物序列 Reverse primer sequences(5'- 3') |
---|---|---|---|---|
Sm18S | Samuk09G0136600 | 221 | AGATTGAAGGCGTTCTTTGGAT | TCAGTGTTTATGGACGGTGGAC |
SmACT | Samuk13G0061200 | 129 | AGAAAGTTGGCCTCGCTGAA | CAGGAACCAGACCACCTGTC |
SmEF-1α | Samuk14G0055700 | 101 | TCCAAGGCCAGGTACGATGA | AAACCAGAGATGGGGACGAAG |
SmRPL1 | Samuk07G0011500 | 206 | CTGACCACCCCACCACTCTC | TCCTCCTCATCTGCCACCTC |
SmRPS26 | Samuk08G0043900 | 230 | GCGAAGAAATGGAGGAAGGA | AGTGGATGGCACACGAAACA |
SmTBCC | Samuk01G0035500 | 130 | AGCCTGACACCCTTTCTCTACC | CCAACCTCTTCTGCAGCCTT |
SmUBC12 | Samuk11G0037500 | 102 | TTGTCTGGACCAACCAAAGGA | TGCTTCTTGCCAGGTGTTTTC |
SmUBP | Samuk09G0089700 | 180 | TGCACAGTTGTTGCTCATGC | CTGCTTCATTTTCCCCGTGC |
内参基因 Reference gene | 基因ID Gene ID | 产物长度 Length of product /bp | 正向引物序列 Forward primer sequences(5'- 3') | 反向引物序列 Reverse primer sequence(5'- 3') |
---|---|---|---|---|
SmAACT4 | Samuk13G0079600 | 132 | CCTGTTTTGAGGGCATTGATTG | ACCAGCATAGAACGCAGCCA |
SmDXS4 | Samuk14G0066700 | 194 | AACAATGCTCAGAAGCCGAGA | GCGATCCATCCAGTAATCCAC |
SmFPS | Samuk04G0156300 | 131 | GAGGCAGAAGTAGGAACTCACCA | CCATTGTAGCAGTCAAGTGGTAAG |
SmbAS1 | Samuk02G0323400 | 113 | GAGTGGGATACTGGTTTCGCTAT | TCCTTGACCTGAGATGCTTTGA |
SmCYP716A-5 | Samuk08G0024600 | 146 | GCTGTTTTCTGTGGCCCTTC | CGAGTCTTGGCAATTTTCCC |
SmUGT73C-14 | Samuk12G0007500 | 120 | ACCTACAAGTGCCCAAACAGAT | ATTCCCAGTTCACCACATTCC |
SmbHLH8 | Samuk10G0068800 | 107 | CAACCTTTGACTCCGACTCCA | CCTTCCCTTACCCTAACCTCC |
SmERF25 | Samuk11G0050000 | 129 | CAACAGCCACACCAACAGCA | TCTTCTCGGGTCACGGATCTC |
Table 2 Primers of objective genes
内参基因 Reference gene | 基因ID Gene ID | 产物长度 Length of product /bp | 正向引物序列 Forward primer sequences(5'- 3') | 反向引物序列 Reverse primer sequence(5'- 3') |
---|---|---|---|---|
SmAACT4 | Samuk13G0079600 | 132 | CCTGTTTTGAGGGCATTGATTG | ACCAGCATAGAACGCAGCCA |
SmDXS4 | Samuk14G0066700 | 194 | AACAATGCTCAGAAGCCGAGA | GCGATCCATCCAGTAATCCAC |
SmFPS | Samuk04G0156300 | 131 | GAGGCAGAAGTAGGAACTCACCA | CCATTGTAGCAGTCAAGTGGTAAG |
SmbAS1 | Samuk02G0323400 | 113 | GAGTGGGATACTGGTTTCGCTAT | TCCTTGACCTGAGATGCTTTGA |
SmCYP716A-5 | Samuk08G0024600 | 146 | GCTGTTTTCTGTGGCCCTTC | CGAGTCTTGGCAATTTTCCC |
SmUGT73C-14 | Samuk12G0007500 | 120 | ACCTACAAGTGCCCAAACAGAT | ATTCCCAGTTCACCACATTCC |
SmbHLH8 | Samuk10G0068800 | 107 | CAACCTTTGACTCCGACTCCA | CCTTCCCTTACCCTAACCTCC |
SmERF25 | Samuk11G0050000 | 129 | CAACAGCCACACCAACAGCA | TCTTCTCGGGTCACGGATCTC |
内参基因 Reference gene | 扩增效率 Amplification efficiency /% | 相关系数R2 Correlation coefficient R2 | 斜率K Slope K |
---|---|---|---|
Sm18S | 97.872 | 0.998 | -3.374 |
SmACT | 93.727 | 0.999 | -3.482 |
SmEF-1α | 99.789 | 0.999 | -3.327 |
SmRPL1 | 100.206 | 0.999 | -3.317 |
SmRPS26 | 94.469 | 0.999 | -3.462 |
SmTBCC | 97.196 | 0.997 | -3.391 |
SmUBC12 | 96.881 | 0.999 | -3.399 |
SmUBP | 98.032 | 0.999 | -3.370 |
Table 3 Amplification efficiency and standard curve para-meters of candidate reference genes
内参基因 Reference gene | 扩增效率 Amplification efficiency /% | 相关系数R2 Correlation coefficient R2 | 斜率K Slope K |
---|---|---|---|
Sm18S | 97.872 | 0.998 | -3.374 |
SmACT | 93.727 | 0.999 | -3.482 |
SmEF-1α | 99.789 | 0.999 | -3.327 |
SmRPL1 | 100.206 | 0.999 | -3.317 |
SmRPS26 | 94.469 | 0.999 | -3.462 |
SmTBCC | 97.196 | 0.997 | -3.391 |
SmUBC12 | 96.881 | 0.999 | -3.399 |
SmUBP | 98.032 | 0.999 | -3.370 |
Fig. 2 Ct values of candidate reference genes The upper and lower parts of the box whiskers indicate the maximum and minimum values of the Ct value;the upper and lower parts of the box body indicate the upper and lower quartiles of the Ct value;lines across the boxes depict the medians;solid diamonds represent outliers and hollow squares represent averages
内参基因 Reference gene | geNorm | NormFinder | BestKeeper | Delta Ct | RefFinder综合排序 Comprehensive ranking | ||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
M值 M value | 排序 Rank | SV值 SV value | 排序 Rank | SD值 SD value | 排序 Rank | CV 值 CV value/% | 排序 Rank | r | 排序 Rank | 平均标准偏差 STDEV | 排序 Rank | 基因稳定性Gene stability | 排序 Rank | ||||||
Sm18S | 0.710 | 6 | 0.722 | 7 | 1.41 | 1 | 5.71 | 4 | 0.950 | 5 | 0.900 | 7 | 4.300 | 5 | |||||
SmACT | 0.373 | 1 | 0.347 | 1 | 1.51 | 4 | 6.04 | 5 | 0.985 | 1 | 0.670 | 1 | 1.410 | 1 | |||||
SmEF-1α | 0.799 | 7 | 0.943 | 8 | 1.54 | 6 | 7.19 | 7 | 0.922 | 6 | 1.070 | 8 | 7.740 | 8 | |||||
SmRPL1 | 0.373 | 1 | 0.373 | 3 | 1.53 | 5 | 5.65 | 3 | 0.985 | 1 | 0.680 | 2 | 2.450 | 2 | |||||
SmRPS26 | 0.656 | 5 | 0.666 | 6 | 1.68 | 7 | 7.65 | 8 | 0.982 | 2 | 0.860 | 6 | 6.450 | 7 | |||||
SmTBCC | 0.615 | 4 | 0.501 | 5 | 1.45 | 3 | 5.18 | 1 | 0.973 | 4 | 0.770 | 5 | 4.400 | 6 | |||||
SmUBC12 | 0.508 | 2 | 0.480 | 4 | 1.53 | 5 | 6.31 | 6 | 0.979 | 3 | 0.740 | 4 | 3.940 | 4 | |||||
SmUBP | 0.547 | 3 | 0.362 | 2 | 1.43 | 2 | 5.59 | 2 | 0.985 | 1 | 0.700 | 3 | 2.630 | 3 |
Table 4 GeNorm,NormFinder and BestKeeper analysis results and rankings
内参基因 Reference gene | geNorm | NormFinder | BestKeeper | Delta Ct | RefFinder综合排序 Comprehensive ranking | ||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
M值 M value | 排序 Rank | SV值 SV value | 排序 Rank | SD值 SD value | 排序 Rank | CV 值 CV value/% | 排序 Rank | r | 排序 Rank | 平均标准偏差 STDEV | 排序 Rank | 基因稳定性Gene stability | 排序 Rank | ||||||
Sm18S | 0.710 | 6 | 0.722 | 7 | 1.41 | 1 | 5.71 | 4 | 0.950 | 5 | 0.900 | 7 | 4.300 | 5 | |||||
SmACT | 0.373 | 1 | 0.347 | 1 | 1.51 | 4 | 6.04 | 5 | 0.985 | 1 | 0.670 | 1 | 1.410 | 1 | |||||
SmEF-1α | 0.799 | 7 | 0.943 | 8 | 1.54 | 6 | 7.19 | 7 | 0.922 | 6 | 1.070 | 8 | 7.740 | 8 | |||||
SmRPL1 | 0.373 | 1 | 0.373 | 3 | 1.53 | 5 | 5.65 | 3 | 0.985 | 1 | 0.680 | 2 | 2.450 | 2 | |||||
SmRPS26 | 0.656 | 5 | 0.666 | 6 | 1.68 | 7 | 7.65 | 8 | 0.982 | 2 | 0.860 | 6 | 6.450 | 7 | |||||
SmTBCC | 0.615 | 4 | 0.501 | 5 | 1.45 | 3 | 5.18 | 1 | 0.973 | 4 | 0.770 | 5 | 4.400 | 6 | |||||
SmUBC12 | 0.508 | 2 | 0.480 | 4 | 1.53 | 5 | 6.31 | 6 | 0.979 | 3 | 0.740 | 4 | 3.940 | 4 | |||||
SmUBP | 0.547 | 3 | 0.362 | 2 | 1.43 | 2 | 5.59 | 2 | 0.985 | 1 | 0.700 | 3 | 2.630 | 3 |
Fig. 4 RT-qPCR expression pattern of genes related to the triterpenoid saponin biosynthesis of S. mukorossi R,root;ST,stem;L,leaf;B,bud;MF,male flower;FF,female flower;S1,pericarp at 15 d after flower pollination;S2,pericarp at 45 d after flower pollination;S3,pericarp at 75 d after flower pollination;S4,pericarp at 90 d after flower pollination;S5,pericarp at 105 d after flower pollination;S6,pericarp at 120 d after flower pollination;S7,pericarp at 135 d after flower pollination;S8,pericarp at 150 d after flower pollination
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