生物技术通报 ›› 2023, Vol. 39 ›› Issue (2): 126-138.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0567
穆德添1(), 万凌云2, 章瑶1, 韦树根2, 陆英1, 付金娥2, 田艺1, 潘丽梅2(), 唐其1()
收稿日期:
2022-05-09
出版日期:
2023-02-26
发布日期:
2023-03-07
作者简介:
穆德添,男,硕士研究生,研究方向:药用植物分子生物学;E-mail: 基金资助:
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
Published:
2023-02-26
Online:
2023-03-07
摘要:
为研究钩藤不同部位中最适管家基因与钩藤生物碱上游合成途径中关键酶基因的表达模式。以钩藤根、茎钩、叶片、蒴果为实验材料,通过RT-qPCR技术、GeNorm、NormFinder、Bestkeeper软件及△Ct程序、RefFinder在线网站分析了12个候选管家基因(18S、SAM、TUA、TUB、EF-1β、EF-1α、RNA L13、GAPDH、Actin6、PAL、CYP、cdc73)表达稳定性。结果表明,最适管家基因为SAM。再以SAM为管家基因,基于“基因组+转录组+代谢组”共表达分析筛选出钩藤生物碱上游合成途径中的15个重点候选相关基因(G8H、8-HGO、IS、CYP76A26、7-DLGT、7-DLH、LAMT、SLS、AS、AnPRT、IGPS、TSA、TSB、TDC、STR)进行表达分析,结果显示这些基因的表达量与含量趋势较为一致,很可能参与钩藤中TIAs的合成。
穆德添, 万凌云, 章瑶, 韦树根, 陆英, 付金娥, 田艺, 潘丽梅, 唐其. 钩藤管家基因筛选及生物碱合成相关基因的表达分析[J]. 生物技术通报, 2023, 39(2): 126-138.
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 |
表1 钩藤生物碱上游合成途径中关键酶基因RT-qPCR的引物序列
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 |
表2 钩藤12个候选管家基因RT-qPCR的引物序列和扩增系数
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 |
图1 钩藤不同部位中钩藤碱(A)和异钩藤碱(B)的含量
Fig. 1 Rhynchophylline and isorhynchophylline contents from U. rhynchophylla different issues *:P<0.01 ;**:P<0.01, the same below
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 |
表3 BestKeeper分析12个候选内参基因表达的稳定性
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 |
图9 异胡豆苷合酶基因筛选热图 Rhynchophylline为钩藤碱含量,Isorhynchophylline为异钩藤碱含量
Fig. 9 Screening heatmap of strictosidine synthase gene Rhynchophylline is the content of rhynchophylline, and isorhynchophylline is the content of isorhynchophylline
图10 以SAM为管家基因时钩藤生物碱生物合成上游途径中关键酶基因表达模式分析
Fig. 10 Expression analysis of upstream pathway genes involved in TIAs biosynthesis using SAM as house-keeping gene
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