Biotechnology Bulletin ›› 2023, Vol. 39 ›› Issue (2): 126-138.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0567
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MU De-tian1(), WAN Ling-yun2, ZHANG Yao1, WEI Shu-gen2, LU Ying1, FU Jin-e2, TIAN Yi1, PAN Li-mei2(), TANG Qi1()
Received:
2022-05-09
Online:
2023-02-26
Published:
2023-03-07
MU De-tian, WAN Ling-yun, ZHANG Yao, WEI Shu-gen, LU Ying, FU Jin-e, TIAN Yi, PAN Li-mei, TANG Qi. House-keeping Genes Screening and Expression Patterns Analysis of Genes Involved in Alkaloid Biosynthesis in Uncaria rhynchophylla[J]. Biotechnology Bulletin, 2023, 39(2): 126-138.
基因Gene | 基因全称Gene name | 引物序列Primer sequence(5'-3') |
---|---|---|
G8H | Geraniol 8-hydroxylase | F:CGCCCATAATCATTCCACTA R:GCCAATGTCACTGCCTCAC |
8-HGO | 8-hydroxygeraniol oxidoreductase | F:GCGTCTGGTGTTCTATCTCCGTT R:GGTTGCGTTGCTGCCTACAT |
IS | Iridoid synthase | F:ACCCTTCAGAAACTCAACAAAAGC R:CCTCGCAACATTCTGGGCTA |
CYP76A26 | Nepetalactol monooxygenase | F:ACCTTATGCTGTCGAGGGATT R:TAACACTGCCAGCTTTCCTGT |
7-DLGT / UGT85A24 | 7-deoxyloganetic acid glucosyltransferase | F:CATGGAAGAAGACAGCAGGAG R:CATTGTCGAATTCCAACCACT |
7-DLH / CYP2A224 | 7-deoxyloganate 7-hydroxylase | F:AGGAACAACAGGATGAGAGCA R:AGGTAGTGTCCTGTCCAGCAA |
LAMT | Loganate methyltransferase | F:TGTCAAAGAAATGCCTGAAGAAG R:ATGCGGAAAGGCTTAGATGG |
SLS / CYP72A1 | Secologanin synthase | F:TCGGACCATTCAAACCTACGG R:GCTTGGACCATCTGTCACCCTC |
AS | Anthranilate synthase | F:TCAAGGACGAAGGGTGGAACAG R:ACCAACCCAACCACCACAAAAT |
AnPRT | Anthranilate phosphoribosyltransferase | F:GTTGGGACTGGTGGTGATGG R:CTTGACCTATTTCCTTGCTTTGC |
IGPS | Indole-3-glycerol phosphate synthase | F:GTTGGGGAGTCTGGGCTTTT R:GAGTCCGGTGATTGCCTTAGTT |
TSA | Tryptophan synthase alpha chain | F:TGTGAAACAAGTTGCTGGATGG R:AGGAGATTTTGCCTCGCCTA |
TSB | Tryptophan synthase beta chain | F:GCTGAGGTTAGGCCAGTTCATT R:TCCACATTAGTCACCCAGTCCC |
TDC | L-tryptophan decarboxylase | F:GGCAGGTATTTTCCCACGCA R:GTTCCCACGGTAGCACAGA |
STR | Strictosidine synthase | F:GCCGATGGTCGGATTCTCAA R:GGCCCAAATAGGCATCAGCA |
Table 1 Primer sequences for key genes in U. rhynchophylla alkaloids upstream synthesis pathway for RT-qPCR
基因Gene | 基因全称Gene name | 引物序列Primer sequence(5'-3') |
---|---|---|
G8H | Geraniol 8-hydroxylase | F:CGCCCATAATCATTCCACTA R:GCCAATGTCACTGCCTCAC |
8-HGO | 8-hydroxygeraniol oxidoreductase | F:GCGTCTGGTGTTCTATCTCCGTT R:GGTTGCGTTGCTGCCTACAT |
IS | Iridoid synthase | F:ACCCTTCAGAAACTCAACAAAAGC R:CCTCGCAACATTCTGGGCTA |
CYP76A26 | Nepetalactol monooxygenase | F:ACCTTATGCTGTCGAGGGATT R:TAACACTGCCAGCTTTCCTGT |
7-DLGT / UGT85A24 | 7-deoxyloganetic acid glucosyltransferase | F:CATGGAAGAAGACAGCAGGAG R:CATTGTCGAATTCCAACCACT |
7-DLH / CYP2A224 | 7-deoxyloganate 7-hydroxylase | F:AGGAACAACAGGATGAGAGCA R:AGGTAGTGTCCTGTCCAGCAA |
LAMT | Loganate methyltransferase | F:TGTCAAAGAAATGCCTGAAGAAG R:ATGCGGAAAGGCTTAGATGG |
SLS / CYP72A1 | Secologanin synthase | F:TCGGACCATTCAAACCTACGG R:GCTTGGACCATCTGTCACCCTC |
AS | Anthranilate synthase | F:TCAAGGACGAAGGGTGGAACAG R:ACCAACCCAACCACCACAAAAT |
AnPRT | Anthranilate phosphoribosyltransferase | F:GTTGGGACTGGTGGTGATGG R:CTTGACCTATTTCCTTGCTTTGC |
IGPS | Indole-3-glycerol phosphate synthase | F:GTTGGGGAGTCTGGGCTTTT R:GAGTCCGGTGATTGCCTTAGTT |
TSA | Tryptophan synthase alpha chain | F:TGTGAAACAAGTTGCTGGATGG R:AGGAGATTTTGCCTCGCCTA |
TSB | Tryptophan synthase beta chain | F:GCTGAGGTTAGGCCAGTTCATT R:TCCACATTAGTCACCCAGTCCC |
TDC | L-tryptophan decarboxylase | F:GGCAGGTATTTTCCCACGCA R:GTTCCCACGGTAGCACAGA |
STR | Strictosidine synthase | F:GCCGATGGTCGGATTCTCAA R:GGCCCAAATAGGCATCAGCA |
基因Gene | 基因全称Gene name | 引物序列Primer sequence(5'-3') | 斜率k | 扩增效率E/% | 相关系数R2 |
---|---|---|---|---|---|
18S | 18S Ribosomal RNA | F:CTTCGGGATCGGAGTAATGA R:GCGGAGTCCTAGAAGCAACA | -3.14 | 0.97 | 0.999 96 |
GAPDH | Glyceraldehyde-3-phosphate dehydrogenase | F:CAGGAACCCAGAGGAGATT R:GCATCCTTACTTGGAGCAG | -3.41 | 0.96 | 0.999 78 |
Actin6 | Actin 6 | F:ACCGAGCGTGGTTATTCTT R:TTCCTGCTGCTTCCATTCC | -3.54 | 0.92 | 0.991 79 |
EF1-β | Elongation factor 1β | F:AAGGCATCCACCAAGAAGA R:AAGGCAACAATGTCACAGC | -3.49 | 0.93 | 0.998 07 |
TUB | β-Tubulin | F:GGAAGTAATCTGCGACGAG R:TGAGAACAGCACGAGGGAC | -3.12 | 1.09 | 0.998 71 |
CYP | Cyclophilin | F:CGAGAAAGGCGTGGGAAAG R:TGAGACCCGTTGGTGTTGG | -3.2 | 1.05 | 0.998 35 |
EF1-α | Elongation factor 1α | F:GAGCGTGAGCGTGGTATTACTAT R:CCAGTGGTGGAGTCAATGATAA | -3.12 | 1.09 | 0.994 50 |
PAL | Phenylalanine ammonialyase | F:ATCGCTGAATCCTCCAATA R:CCACCCTACTCCACAATACTT | -3.12 | 1.09 | 0.998 68 |
RNA L13 | Ribosomal protein L13 | F:CCAGGAGAAGGAATGCGAGG R:GACCGGTTTTTACGGCGATG | -3.67 | 0.87 | 0.991 99 |
SAM | S-adenosylmethionine decarboxylase | F:CACAATCTGGCATACGAAA R:AACTCACTTGGCTGGAAAC | -3.7 | 0.92 | 0.995 17 |
cdc73 | Cell division control protein 73 | F:TGGTGGCTGTTTTCGTGTT R:TGATGCCGCTTATTCTTGC | -3.44 | 0.95 | 0.990 33 |
TUA | α-Tubulin | F:TCCCTTCTTGAGCACACTGAT R:CCATCAAACCTCAAAGACGCA | -3.28 | 1.02 | 0.993 96 |
Table 2 Primer sequence and amplification parameters for 12 candidate reference genes of U. rhynchophylla in RT-qPCR
基因Gene | 基因全称Gene name | 引物序列Primer sequence(5'-3') | 斜率k | 扩增效率E/% | 相关系数R2 |
---|---|---|---|---|---|
18S | 18S Ribosomal RNA | F:CTTCGGGATCGGAGTAATGA R:GCGGAGTCCTAGAAGCAACA | -3.14 | 0.97 | 0.999 96 |
GAPDH | Glyceraldehyde-3-phosphate dehydrogenase | F:CAGGAACCCAGAGGAGATT R:GCATCCTTACTTGGAGCAG | -3.41 | 0.96 | 0.999 78 |
Actin6 | Actin 6 | F:ACCGAGCGTGGTTATTCTT R:TTCCTGCTGCTTCCATTCC | -3.54 | 0.92 | 0.991 79 |
EF1-β | Elongation factor 1β | F:AAGGCATCCACCAAGAAGA R:AAGGCAACAATGTCACAGC | -3.49 | 0.93 | 0.998 07 |
TUB | β-Tubulin | F:GGAAGTAATCTGCGACGAG R:TGAGAACAGCACGAGGGAC | -3.12 | 1.09 | 0.998 71 |
CYP | Cyclophilin | F:CGAGAAAGGCGTGGGAAAG R:TGAGACCCGTTGGTGTTGG | -3.2 | 1.05 | 0.998 35 |
EF1-α | Elongation factor 1α | F:GAGCGTGAGCGTGGTATTACTAT R:CCAGTGGTGGAGTCAATGATAA | -3.12 | 1.09 | 0.994 50 |
PAL | Phenylalanine ammonialyase | F:ATCGCTGAATCCTCCAATA R:CCACCCTACTCCACAATACTT | -3.12 | 1.09 | 0.998 68 |
RNA L13 | Ribosomal protein L13 | F:CCAGGAGAAGGAATGCGAGG R:GACCGGTTTTTACGGCGATG | -3.67 | 0.87 | 0.991 99 |
SAM | S-adenosylmethionine decarboxylase | F:CACAATCTGGCATACGAAA R:AACTCACTTGGCTGGAAAC | -3.7 | 0.92 | 0.995 17 |
cdc73 | Cell division control protein 73 | F:TGGTGGCTGTTTTCGTGTT R:TGATGCCGCTTATTCTTGC | -3.44 | 0.95 | 0.990 33 |
TUA | α-Tubulin | F:TCCCTTCTTGAGCACACTGAT R:CCATCAAACCTCAAAGACGCA | -3.28 | 1.02 | 0.993 96 |
基因 Gene | 几何平均数 GM | 算术平均数 AM | 最小值 Min | 最大值 Max | 标准偏差 Standard deviation | 变异系数 Coefficient of variation | 稳定性排序 Stability rank |
---|---|---|---|---|---|---|---|
18S | 15.55 | 15.55 | 15.09 | 16.15 | 0.32 | 2.05 | 1 |
SAM | 22.14 | 22.15 | 21.10 | 23.05 | 0.54 | 2.44 | 2 |
CYP | 19.39 | 19.41 | 18.05 | 20.77 | 0.69 | 3.56 | 3 |
EF-1β | 21.58 | 21.59 | 20.43 | 22.68 | 0.75 | 3.48 | 4 |
cdc73 | 24.46 | 24.48 | 23.35 | 26.36 | 0.77 | 3.15 | 5 |
TUB | 24.30 | 24.31 | 23.42 | 25.68 | 0.78 | 3.22 | 6 |
EF-1α | 21.05 | 21.07 | 19.34 | 22.51 | 0.87 | 4.12 | 7 |
PAL | 22.94 | 22.96 | 21.20 | 23.91 | 0.96 | 4.16 | 8 |
Actin6 | 23.46 | 23.51 | 21.11 | 24.90 | 1.17 | 4.91 | 9 |
RNA L13 | 22.50 | 22.54 | 21.03 | 25.07 | 1.18 | 5.21 | 10 |
GAPDH | 26.82 | 26.87 | 24.55 | 28.70 | 1.53 | 5.69 | 11 |
TUA | 22.36 | 22.53 | 19.15 | 26.03 | 2.78 | 12.34 | 12 |
Table 3 Expression stabilities of 12 candidate reference genes analyzed by BestKeeper
基因 Gene | 几何平均数 GM | 算术平均数 AM | 最小值 Min | 最大值 Max | 标准偏差 Standard deviation | 变异系数 Coefficient of variation | 稳定性排序 Stability rank |
---|---|---|---|---|---|---|---|
18S | 15.55 | 15.55 | 15.09 | 16.15 | 0.32 | 2.05 | 1 |
SAM | 22.14 | 22.15 | 21.10 | 23.05 | 0.54 | 2.44 | 2 |
CYP | 19.39 | 19.41 | 18.05 | 20.77 | 0.69 | 3.56 | 3 |
EF-1β | 21.58 | 21.59 | 20.43 | 22.68 | 0.75 | 3.48 | 4 |
cdc73 | 24.46 | 24.48 | 23.35 | 26.36 | 0.77 | 3.15 | 5 |
TUB | 24.30 | 24.31 | 23.42 | 25.68 | 0.78 | 3.22 | 6 |
EF-1α | 21.05 | 21.07 | 19.34 | 22.51 | 0.87 | 4.12 | 7 |
PAL | 22.94 | 22.96 | 21.20 | 23.91 | 0.96 | 4.16 | 8 |
Actin6 | 23.46 | 23.51 | 21.11 | 24.90 | 1.17 | 4.91 | 9 |
RNA L13 | 22.50 | 22.54 | 21.03 | 25.07 | 1.18 | 5.21 | 10 |
GAPDH | 26.82 | 26.87 | 24.55 | 28.70 | 1.53 | 5.69 | 11 |
TUA | 22.36 | 22.53 | 19.15 | 26.03 | 2.78 | 12.34 | 12 |
Fig. 9 Screening heatmap of strictosidine synthase gene Rhynchophylline is the content of rhynchophylline, and isorhynchophylline is the content of isorhynchophylline
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