基于转录组测序的苦瓜皂苷合成相关基因的鉴定和分析

doi:10.3864/j.issn.0578-1752.2024.09.012

Abstract

Abstract:

【Objective】As the main source of bitterness in bitter gourd fruit, saponin possesses various medicinal values including hypoglycemic and anticancer properties. This paper aimed at identifying the metabolic pathways and genes involved in the regulation of bitter gourd saponin biosynthesis, so as to provide the theoretical basis for further analysis of the molecular mechanism of bitter gourd saponin formation.【Method】Using the bitter gourd high-generation inbred line GK24 as the test material, fruit tissues were sampled at the ovary stage (T1), young fruit stage (T2), commodity fruit stage (T3), and maturity fruit stage (T4). The saponin content of bitter gourd at different periods were determined by the vanillin-glacial acetic acid method, and the differentially expressed genes (DEGs) were identified using the method of transcriptome sequencing. 【Result】From T1 to T4, the endogenous saponin content of bitter gourd showed significant decrease with fruit development. A total of 17 504 genes were identified by transcriptome sequencing, and the number of down-regulated genes was higher than that of up-regulated genes in the comparison of the three groups of T1-vs-T2, T2-vs-T3, and T3-vs-T4. GO enrichment analysis showed that phosphorus-containing complex metabolic process, kinesin complex and 2-succinyl-6-hydroxy-cyclohexadiene-1-carboxylic acid synthetase activity were the most significantly enriched terms in biological process, cellular component and molecular function, respectively. KEGG analysis showed that global and overview mapping, carbohydrate metabolism, and amino acid metabolism were the most significantly enriched pathways in all three comparative groups. The genes could be categorized into 20 profiles based on their expression patterns. Genes in profile 0 gradually decreased from T1 to T4, showing similar pattern of saponin content changes with bitter gourd fruit development. 14 genes related to bitter gourd saponin biosynthesis were identified from profile 0, including AAT1, HMG1, MVK, PMK, MVD2, and FPS1 in the terpene skeleton biosynthesis pathway, SS12, SQE1, and CPQ in the sesquiterpene and triterpene biosynthesis pathway, and the postmodifying enzyme genes CYP97A3, CYP71AN24, UGT94E5 and UGT73C6. The results of RNA-seq were validated by qRT-PCR method.【Conclusion】The saponin content of bitter gourd decreased with the fruit development. Bitter gourd saponin was synthesized through the terpenoid bone biosynthesis (ko00900) metabolic pathway to accumulate terpenoid saponin skeleton in the first, and then produce saponins through the sesquiterpene and triterpene biosynthesis (ko00909) metabolic pathway and the modification of oxidoreductase and glycosyltransferase successively. A total of 14 genes related to the saponin biosynthesis of bitter gourd have been identified in the present study.

Key words: bitter gourd, transcriptome, differentially expressed genes, saponin, regulation of synthesis

QI RenJie, NING Yu, LIU Jing, LIU ZhiYang, XU Hai, LUO ZhiDan, CHEN LongZheng. Identification and Analysis of Genes Related to Bitter Gourd Saponin Synthesis Based on Transcriptome Sequencing[J].Scientia Agricultura Sinica, 2024, 57(9): 1779-1793.

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Figures/Tables 12

Table 1

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Table 2

Fig. 2

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Table 3

Table 4

Genes enriched in the ko00900 and ko00909 pathways in profile 0"

基因编号 Gene number	基因注释 Gene function	通路 Pathway	T1 FPKM	T2 FPKM	T3 FPKM	T4 FPKM
evm.TU.chr1.608	法尼基半胱氨酸裂解酶异构体 Farnesylcysteine lyase isoform	ko00900	38.15	28.42	17.24	10.65
evm.TU.chr10.805	2-甲基-D-赤藓糖醇2,4-环二磷酸合酶 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase	ko00900	115.94	71.18	43.36	26.32
evm.TU.chr2.34	香叶基香叶基二磷酸还原酶样异构体 Geranylgeranyl diphosphate reductase-like isoform	ko00900	30.58	17.55	16.44	7.16
evm.TU.chr3.1930	羟基-3-甲基丁-2-烯-1-基二磷酸合酶 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase	ko00900	433.17	194.37	106.87	85.58
evm.TU.chr3.2317	法尼基焦磷酸合酶1 Farnesyl pyrophosphate synthase 1	ko00900	358.50	164.00	51.39	7.70
evm.TU.chr4.352	乙酰辅酶A乙酰转移酶 Acetyl-CoA acetyltransferae	ko00900	439.08	95.24	57.25	34.89
evm.TU.chr4.613	4-羟基-3-甲基丁-2-烯基二磷酸还原酶 4-hydroxy-3-methylbut-2-enyl diphosphate reductase	ko00900	1091.57	439.96	370.39	217.45
evm.TU.chr6.140	1-脱氧-D-酮糖5-磷酸还原异构酶 1-deoxy-D-xylulose 5-phosphate reductoisomerase	ko00900	239.29	131.64	82.92	76.05
evm.TU.chr6.518	脱氧-D-酮糖-5-磷酸合成酶Deoxy-D-xylulose-5-phosphate synthase	ko00900	637.07	42.68	19.10	0.17
evm.TU.chr4.378	HMG-CoA合成酶 3-hydroxy-3-methyl glutaryl coenzyme A synthesis	ko00900	1274.61	1010.52	91.31	2.87
evm.TU.chr8.4201	甲羟戊酸激酶 Mevalonate kinase	ko00900	105.59	52.77	31.46	14.07
evm.TU.chr9.3672	焦磷酸甲羟戊酸脱羧酶Pyrophosphomethylvalerate decarboxylase	ko00900	671.59	131.25	53.80	28.02
evm.TU.chr10.2918	磷酸甲羟戊酸激酶 Phosphomevalonate kinase	ko00900	85.10	58.17	9.86	3.75
evm.TU.chr6.363	磷酸甲羟戊酸激酶 Phosphomevalonate kinase	ko00900	41.95	20.49	13.08	12.38
evm.TU.chr2.1021	角鲨烯合成酶 Squalene synthase	ko00909	437.66	285.81	89.01	48.19
evm.TU.chr3.2797	橙花内酯合成酶 Nerolidol synthase	ko00909	15.85	9.29	0.69	0.07
evm.TU.chr7.10	葫芦二烯醇合酶 Cucurbitadienol synthase	ko00909	981.27	444.87	79.12	4.09
evm.TU.chr8.588	角鲨烯环氧酶 Squalene epoxidase 1	ko00909	1030.32	506.37	76.82	1.14

Table 4

Fig. 7

Fig. 8

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基因名 Gene name	正向引物Forward primer (5′-3′)	反向引物Reverse primer (5′-3′)
CPQ	GTTGAAAGAGGGGGAAGAGG	CACCAGACCCGGAAGTAAAA
FSP1	GCGGATCTCAGGTCAACATT	TCCAGGCACATTGTAGTCCA
CYP97A3	GGGAGATATGGCGTGTAAGAAGAC	CTAAAGTCAGACGAGAGAACAGGG
CYP71AN24	GAACACAAGGCCAAGATGAGAG	CAGTTGTGTCAGTTCCAGCTAC
UGT73C6	TGCCTCCAGAGCTGAAACAT	TCCCATGAATCCAGCCACTT
UGT94E5	TCATCACACAACCAAAGGCC	ATCGGAATGTTGAGCGATGC
SQE1	AGCAAGGGACGGTTACTTCA	TGAGAAGCAGCCATCACAGA
MVK	GCAGTCGTTCATGGATCCAC	TGGCCACGAAAACTCCAATG
PMK	GGGACCATGAGAGGACTGAG	AGGGTCAGACTTTTGCCACT
Actin	GGCAAACCCTAAAGTTTTCTTCG	GATGAGCCCTTGTAATGAAGTGG

样品 Sample	原始数据 Raw reads	过滤后数据 Clean reads	Q30含量 Q30 content (%)	GC含量 GC content (%)	单一比对率 Unique mapped (%)	总比对率 Total mapped (%)
T1-1	46878594	46673674	92.28	46.80	93.74	96.88
T1-2	55531244	55302940	92.51	46.59	93.74	96.89
T1-3	65068774	64756962	91.96	46.30	93.49	96.61
T2-1	53923956	53678084	91.97	46.70	93.42	96.63
T2-2	46863574	46589694	92.55	47.09	93.61	96.87
T2-3	51548340	51304488	92.54	47.04	93.43	96.74
T3-1	44171240	43942622	92.37	46.84	93.10	95.88
T3-2	44849744	44668862	92.68	47.20	94.17	97.07
T3-3	55212924	54973380	92.20	47.14	93.68	96.43
T4-1	69720780	69450220	92.42	47.30	93.73	96.46
T4-2	66405070	66157562	92.94	46.67	94.16%	96.82
T4-3	64248114	63979034	92.29	46.69	92.75	96.29

通路名称 Pathway name	通路ID Pathway ID	基因数目 Number of genes	所有基因数 Number of all genes	P值 P value
核糖体 Ribosome	ko03010	83	305	3.36E-13
同源重组 Homologous recombination	ko03440	20	58	1.03E-05
DNA复制 DNA replication	ko03030	16	46	7.03E-05
氨基酸的生物合成 Biosynthesis of amino acids	ko01230	44	202	9.41E-05
错配修复 Mismatch repair	ko03430	14	39	0.000135124
缬氨酸、亮氨酸和异亮氨酸生物合成 Valine, leucine and isoleucine biosynthesis	ko00290	8	18	0.000748137
真核生物核糖体的生物合成 Ribosome biogenesis in eukaryotes	ko03008	19	77	0.002133956
嘧啶代谢 Pyrimidine metabolism	ko00240	15	58	0.00377061
2-氧代羧酸代谢 2-Oxocarboxylic acid metabolism	ko01210	14	53	0.004117066
糖鞘脂生物合成 Glycosphingolipid biosynthesis	ko00604	3	4	0.006896205
精氨酸生物合成 Arginine biosynthesis	ko00220	10	35	0.008097428
碱基切除修复 Base excision repair	ko03410	13	52	0.009192653
核质运输 Nucleocytoplasmic transport	ko03013	20	98	0.01563779
赖氨酸生物合成 Lysine biosynthesis	ko00300	5	13	0.01582533
萜类骨架生物合成 Terpenoid backbone biosynthesis	ko00900	14	62	0.0173777
糖胺聚糖降解 Glycosaminoglycan degradation	ko00531	6	18	0.01771733
氨酰基tRNA生物合成 Aminoacyl-tRNA biosynthesis	ko00970	13	57	0.0198342
不同类型的N-聚糖生物合成 Various types of N-glycan biosynthesis	ko00513	8	30	0.02599519
组氨酸代谢 Histidine metabolism	ko00340	5	16	0.03922549

Identification and Analysis of Genes Related to Bitter Gourd Saponin Synthesis Based on Transcriptome Sequencing

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