钩藤管家基因筛选及生物碱合成相关基因的表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2022-0567

摘要/Abstract

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。

关键词: 钩藤, 实时荧光定量PCR, 管家基因, 吲哚萜类生物碱, 关键酶基因, 表达

Abstract:

This work aims to study the expression pattern of the most suitable house-keeping genes in different parts of Uncaria rhynchophylla and the key enzymes genes in the upstream synthesis pathway of U. rhynchophylla indole alkaloids. The root, stem hook, leaf and capulse from U. rhynchophylla were used as experimental materials. The expression stabilities of 12 candidate house-keeping genes（18S, SAM, TUA, TUB, EF-1β, EF-1α, RNA L13, GAPDH, Actin6, PAL, CYP, and cdc73）were evaluated by RT-qPCR, GeNorm, NormFinder, Bestkeeper, △Ct, and RefFinder. The results showed that SAM can be used as the most suitable house-keeping genes for gene expression analysis in U. rhynchophylla. Having SAM as house-keeping gene, 15 key enzyme genes involved in alkaloid biosynthesis upstream pathway, i.e., G8H, 8-HGO, IS, CYP76A26, 7-DLGT, 7-DLH, LAMT, SLS, AS, AnPRT, IGPS, TSA, TSB, TDC, and STR were screened based on the co-expression analysis of “genome + transcriptome + metabolome”, and their expression levels were analyzed. The results showed that the expressions of these genes were consistent with the content trend, indicating that the genes are likely involved in the synthesis of terpenoid indole alkaloids.

Key words: Uncaria rhynchophylla, RT-qPCR, house-keeping gene, terpenoid indole alkaloids, gene of key enzyme, expression

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.

图/表 13

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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'）
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

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