Transcriptome Analysis of Ardisia crenata Sims Induced by Salicylic Acid and Key Enzyme Gene Mining in Triterpenoid Saponin Biosynthesis Pathway

doi:10.7668/hbnxb.20193579

Abstract

Abstract:

Ardisia crenata Sims is a traditional Chinese medicine plant belonging to Myrsinaceae family.The oleanane-type triterpenoid saponin is regarded as the main active ingredient accumulated in the root.In order to explore the molecular basis of the biosynthesis of triterpenoid saponins,the Illumina HiseqTM 4000 high throughput sequencing method was used to analyze the transcriptome of 3-year-old plants of A.crenata treated with sterile water and 2 mmol/L SA.The transcriptome data of roots were obtained,and GO classification,KOG functional annotation,KEGG metabolic pathway analysis,protein function annotation analysis,differential gene analysis and discovery of key genes in metabolic pathways were completed based on Blast.A total of 41.63 Gb Clean data were obtained,102 491 Unigenes were assembled by de novo splicing with an average length of 831 bp,and the annotation rate was 50.21%.Through differential analysis of genes responding to SA,a total of 3 417 differentially expressed genes(DEGs)were found,of which 2 725 were up-regulated and 692 were down-regulated.DEGs were significantly enriched in secondary metabolite biosynthesis,metabolic pathways,amino and nucleotide metabolism,Glycolysis/Gluconeogenesis,carbon metabolism pathways.Metabolic pathways related to triterpenoid saponin in A.crenata included the pathways of terpene skeleton biosynthesis(ko00900)and sesquiterpene and triterpene biosynthesis(ko00909).A total of 8 key enzymes and 11 differentially expressed Unigenes were screened.Under salicylic acid treatment, the differential expression of HMGR, HMGS, MVK, GGPS, SS, and SQE genes was significant in MVA pathway of terpene skeleton biosynthesis, while the differential expression of DXS, DXR, MCT, CMK, MDS, and HDR genes in MEP pathway was not significant. The gene expression of beta-amyrin synthase(β-AS),the key enzyme that catalyzes the formation of oleanolane pentacyclic triterpene precursors,was down-regulated,as was the expression of the post-modification genes CYP450s(CYP72A67).The change trend of qPCR value was consistent with that of FPKM value of RNA-seq.It is speculated that SA affected the accumulation of triterpenoid saponins by inducing the expression of key enzyme genes in the MVA pathway,and the enzyme genes in MEP pathway may participate in the synthesis of secondary metabolites in other pathways.

Key words: Ardisia crenata Sims, Triterpenoid saponins, Transcriptome, Salicylic acid, Differential expression, qPCR

LU Yan, MA Mingdong, CAO Hui, LI Yuanyuan. Transcriptome Analysis of Ardisia crenata Sims Induced by Salicylic Acid and Key Enzyme Gene Mining in Triterpenoid Saponin Biosynthesis Pathway[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(2): 106-119. doi: 10.7668/hbnxb.20193579.

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Figures/Tables 18

Tab.1 Sequences of primers used for qPCR

基因编号 Unigene ID	基因名 Gene name	正向引物(5'-3') Forward primer(5'-3')	反向引物(5'-3') Reverse primer(5'-3')	扩增长度/bp Amplification length
0029023	Actin 1	ACGGAGAGAGAGAGCCAGTC	GCATCATCACCAGCAAACCC	170
0039038	MYB48	TAACCTCGTTTGGGTCGTGT	GGGGTGTCATCTTGCCTCG	129
0030526	SBE1	GACCCACCACCCTCAGAAAG	GCCATCAACTGGACCGTG	189
0101965	SPS1	ATGAAGGGCTCTCAAAAGGC	TGTTTCCTCCTCCTCAATGCC	86
0029224	CYP72A67	AGAAATGAAGGAGGGGCGAG	CCCAGCAAAACCAGTGTCC	201
0039040	MYB48	GCTGCGTTGGGTGAACTATC	TGAGCCTTCTTCCTCGTGTG	442
0056938	HOMT1	GGTGGCAAAGAGAGGACTGA	GGAGCACATCACTCGGAAC	81
0076745	SQE1	CCCCTTATCCTACACCTGGC	TAGATGCCACAGGCTTTCGT	204
0083134	CYP94A2	AGAAATCCTCCGAAACACACC	ACCGTCACCAAGCAAGTCAG	175
0075409	β-AS	CCCGCATCCCATCCTACAAG	TCCGCCCAACAAGAAAGCAA	204

Fig.1 Triterpene saponin contents of A. crenata with different treatments

Tab.2 The RNA sample quality information

样品 Sample	浓度/(ng/μL) Concentration	体积/μL Volume	总含量/μg Total content	OD_260/280	OD_260/230	RIN值 RIN value	28S/18S
空白CK	55	46	2.53	2.03	2.07	8.2	2.12
处理Treatment	83	42	3.49	2.10	2.02	8.4	2.20

Fig.2 Length distribution of Unigenes

Tab.3 Annotation statistics of Unigenes

序号 Serial number	注释数据库 Annotated databases	注释基因数量 Number of annotated Unigenes	注释率/% Annotation percentage
1	Nr	44 789	43.70
2	Swiss Prot	42 331	41.30
3	KEGG	40 151	39.18
4	KOG	33 059	32.26
5	GO	34 628	33.79
6	Pfam	29 382	28.67
有注释Annotated		51 457	50.21
总计Total		102 491	100.00

Fig.3 Distribution map of homologous sequence species in Nr database

Fig.4 Gene ontology functional classifications of Unigenes in A.crenata 1.Cellular process;2.Metabolic process;3.Single-organism process;4.Response to stimulus;5.Biological regulation;6.Regulation of biological process;7.Localization;8.Cellular component organization or biogenesis;9.Developmental process;10.Multicellular organismal process; 11.Signaling;12.Multi-organism process;13.Reproduction;14.Reproductive process;15.Negative regulation of biological process; 16.Positive regulation of biological process;17.immune system process;18.Growth;19.Locomotion;20.Detoxification;21.Biological adhesion;22.Rhythmic process;23.Behavior; 24.Cell aggregation;25.Presynaptic process involved in synaptic transmission;26.Cell killing;27.Cell;28.Cell part;29.Organelle; 30.Membrane; 31.Macromolecular complex; 32.0rganelle part;33.Membrane part;34.Membrane-enclosedlumen;35.Extracellular region;36.Cell junction;37.Symplast; 38.Supramolecular fiber;39.Extracellular region part;40.Virion;41.Virion part;42.Synapse;43.Other organism;44.Other organism part;45.Nucleoid; 46.Synapse part; 47.Extracellular matrix;48.Viral occlusion body;49.Extracellular matrix component;50.Binding;51.Catalytic activity; 52.Transporter activity; 53.Structural molecule activity;54.Nucleic acid binding transcription factor activity;55.Molecular function regulator;56.Electron carrier activity; 57.Signal transducer activity;58.Antioxidant activity;59.Molecular transducer activity;60.Transcription factor activity,protein binding;61.Translation regulator activity; 62.Metallochaperone activity;63.Nutrient reservoir activity; 64.Protein tag;65.Chemoattractant activity.

Fig.5 KOG function classification of Unigene in A.crenata 1.Translation,ribosomal structureandbiogenesis;2.RNA processing and modification;3.Transcription; 4.Replication,recombination and repair;5.Chromatin structure and dynamics;6.Cell cycle control, cell division, chromosome partitioning;7.Nuclear structure;8.Defense mechanisms;9.Signal transduction mechanisms; 10.Cell wall/membrane/envelope biogenesis;11.Cell motility;12.Cytoskeleton;13.Extracellular structures;14.Intracellular trafficking, secretion, and vesicular transport;15.Posttranslational modification,protein turnover,chaperones; 16.Energy production and conversion;17.Carbohydrate transport and metabolism;18.Amino acid transport and metabolism; 19.Nucleotide transport and metabolism;20.Coenzyme transport and metabolism;21.Lipidtransport and metabolism; 22.Inorganicion transport and metabolism;23.Secondary metabolites biosynthesis transport and catabolism; 24.General function prediction only;25.Function unknown.

Fig.6 KEGG metabolic pathway classification of Unigenes in A.crenata

Tab.4 Statistics of KEGG standard metabolic pathways(top 20)

编号 No.	KEGG代谢通路 KEGG Pathway	基因数量 Unigene number	通路号 Pathway ID
1	代谢通路	6 354	ko01100
2	次生代谢物生物合成	3 191	ko01110
3	核糖体	1 503	ko03010
4	碳代谢	1 180	ko01200
5	氨基酸生物合成	856	ko01230
6	氧化磷酸化	736	ko00190
7	内质网蛋白加工	735	ko04141
8	RNA转运	633	ko03013
9	剪接体	590	ko03040
10	糖酵解/糖异生	565	ko00010
11	内吞作用	551	ko04144
12	氨基酸和核苷酸代谢	440	ko00520
13	淀粉和蔗糖代谢	423	ko00500
14	丙酮酸代谢	411	ko00620
15	柠檬酸循环	403	ko00020
16	吞噬体	388	ko04145
17	嘌呤代谢	374	ko00230
18	半胱氨酸和蛋氨酸代谢	371	ko00270
19	脂肪酸代谢	358	ko01212
20	信使RNA监测通路	349	ko03015

Fig.7 Hierarchical clustering heat map of DEGs

Fig.8 Classification of GO functions of DEGs 1.Cellular process;2.Metabolic process;3.Single-organism process;4.Response to stimulus;5.Biological regulation;6.Regulation of biological process;7.Localization;8.Cellular component organization or biogenesis;9.Mult-organism process;10.Developmental process;11.Signaling; 12.Multicellular organismal process;13.Reproduction;14.Negative regulation of biological process;15.Reproductive process;16.Positive regulation of biological process; 17.Lmmune system process;18.Detoxification;19.Locomotion;20.Growth;21.Biological adhesion;22.Presynaptic process involved in synaptic transmission; 23 Rhythmic process;24.Behavior;25.Cell aggregation;26.Cell;27.Cell part;28.Membrane;29.Organelle;30.Macromolecuar complex; 31.Membrane part; 32.Organelle part;33.Extracellular region;34.Membrane-enclosed lumen;35.Cell junction;36.Symplast;37.Supramolecular fiber;38.Virion;39.Extracellular region part;40.Virion part;41.Extracellular matrix;42.Synapse part;43.Nucleoid;44.Synapse;45.Catalytic activity;46.Binding;47.Transporter activity; 48.Structural molecule activity;49.Nucleic acid binding transcription factor activity;50.Electron carrier activity;51.Molecular function regulator; 52.Antioxidant activity; 53.Molecular transducer activity; 54.Signal transducer activity;55.Transcription factor activity protein binding;56.Metallchaperone activity;57.Nutrient reservoir activity.

Fig.9 KEGG metabolic pathway analysis of DEGs(Top 20) 1.Biosynthesis of secondary metabolites;2.Metabolic pathways;3.Amino sugar and nucleotide sugar metabolism;4.Glycolysis/Gluconeogenesis;5.Carbon metabolism;6.Glyoxylate and dicarboxylate metabolism;7.Biosynthesis of amino acids;8.Cysteine and methionine metabolism;9.Phenylpropanoid biosynthesis;10.Diterpenoid biosynthesis;11.Fatty acid degradation;12.Limonene and pinene degradation;13.Carbon fixation in photosynthetic organisms;14.Plant hormone signal transduction;15.Linoleic acid metabolism;16.Sesquiterpenoid and triterpenoid biosynthesis;17.Starch and sucrose metabolism;18.Citrate cycle(TCA cycle);19.Zeatin biosynthesis; 20.Alanine, aspartate and glutamate metabolism.

Tab.5 Genes involved in triterpenoid biosynthesis in A. crenata

编号 Number	名称 Name	简称 Abbreviation	K编号 K ID	单基因数量 Number of Unigenes
1	乙酰辅酶A酰基转移酶	AACT	K00626	21
2	羟甲基戊二酰辅酶A合成酶	HMGS	K01641	14
3	羟甲基戊二酰辅酶A还原酶	HMGR	K00021	19
4	甲羟戊酸激酶	MVK	K00869	3
5	磷酸甲羟戊酸激酶	PMK	K00938	1
6	甲羟戊酸-5-二磷酸脱羧酶	MPD	K01597	3
7	1-脱氧-D-木糖-5-磷酸合酶	DXS	K01662	4
8	1-脱氧-D-木糖-5-磷酸还原异构酶	DXR	K00099	1
9	2-C-甲基-D-赤藓糖醇-4-磷酸胞苷转移酶	MCT	K00991	1
10	4-(胞苷-5-二磷酸)-2-C-甲基赤藓糖醇激酶	CMK	K00919	1
11	2-C-甲基赤藓醇-2,4-环二磷酸合酶	MCS	K01770	1
12	4-羟基-3-甲基-丁基-2-苯基二磷酸还原酶	HDR	K03527	1
13	异戊烯基二磷酸异构酶	IDI	K01823	6
14	香叶基焦磷酸合成酶	GPS	K14066	1
15	香叶基香叶基二磷酸合酶	GGPPS	K13789	6
16	法尼基二磷酸合酶	FPS	K00787	5
17	鲨烯合酶	SS	K00801	9
18	鲨烯环氧酶	SQE	K00511	8
19	β-香树素合成酶	β-AS	K15813	4
20	细胞色素P450	CYP72A67	K15472	1

Tab.6 DEGs of triterpenoid metabolism pathway in A. crenata

编号 Number	基因号 Gene ID	FPKM值 FPKM value		简称 Abbreviation	基因名称 Gene name
编号 Number	基因号 Gene ID	CK	T	简称 Abbreviation	基因名称 Gene name
1	Unigene0076745	67.12	11.97	SQE	鲨烯环氧酶
2	Unigene0049294	12.37	2.54	SQE	鲨烯环氧酶
3	Unigene0053097	4.25	0.36	SQE	鲨烯环氧酶
4	Unigene0058867	7.42	1.84	SS	鲨烯合酶
5	Unigene0080472	7.98	1.24	HMGR	3-羟基-3-甲基戊二酰辅酶
6	Unigene0080469	7.40	1.79	HMGR2	3-羟基-3-甲基戊二酰辅酶
7	Unigene0029224	55.42	9.13	CYP72A67	细胞色素P450
8	Unigene0075409	46.44	6.64	β-AS	β-香树素合成酶
9	Unigene0045058	2.47	0.20	HMGS	羟甲基戊二酰辅酶A合成酶
10	Unigene0005438	20.23	9.67	MVK	甲羟戊酸激酶
11	Unigene0038713	10.08	3.49	GPS	香叶基焦磷酸合成酶

Fig.10 Verification of RNA-seq data by qPCR in A. crenata Different letters indicate significant difference at the level of P<0.05.

Tab.7 FPKM values of 9 RNA-seq genes

编号 Number	基因号 Gene ID	符号 Symbol	FPKM值 FPKM value		上调/下调 Up/Down
编号 Number	基因号 Gene ID	符号 Symbol	CK	T	上调/下调 Up/Down
1	Unigene0039038	MYB48	361.38	134.44	下调
2	Unigene0030526	SBE1	293.02	96.91
3	Unigene0101965	SPS1	259.17	104.48
4	Unigene0029224	CYP72A67	55.42	9.13
5	Unigene0039040	MYB48	141.02	66.99
6	Unigene0076745	SQE	67.12	11.97
7	Unigene0075409	β-AS	46.44	6.64
8	Unigene0056938	HOMT1	85.60	175.05	上调
9	Unigene0083134	CYP94A2	65.95	271.27

Fig.11 Speculated diagram of synthetic pathway of triterpenoid saponins in A. crenata AACT.Acetyl-CoA acyltransferase;HMGS.3-hydroxy-3-methylglutaryl CoA synthetase;HMGR.3-hydroxy-3-methylglutary1 CoA reductase; MVK.Mevalonate kinase PMK.Phosphomevalonate kinase; MVD.5-mevalonate pyrophosphate decarboxylase;IPP.Isopentenyl pyrophosphate;IDI.Isopentenyl pyrophosphate isomerase; DMAPP.Dimethy1ally1 pyrophosphate;DXS.1-deoxy-D-xylulose-5-phosphate synthetase;DXR.1-deoxy-D-xylulose-5-phosphate reductoisomerase; MCT.Cytidine 4- diphosphate-2-C-methyl-D-erythritol synthetase; CMK.Cytidine 4-diphosphate 2-C-methyl-D-erythritol kinase;MDS.2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthetase;HDS.(E)-4-hydroxy-3-methy1-2-butenyl pyrophosphate synthetase;HDR.(E)-4-hydroxy-3-methyl-2-buteny1 pyrophosphate reductase; GPS.Gerany pyrophosphate synthetase; FPS.Farnesyl pyrophosphate synthetase;SS.Squalene synthetase; SQE.Squalene oxidase;β-AS.β-amyrin synthetase; CYP450.Cytochrome P450;UGT.Glycosyltransferase.Dashed arrows indicate multiple steps.

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