早熟砂梨‘苏翠1号’与其亲本‘翠冠’‘华酥’成熟果实差异代谢产物及差异基因比较分析

doi:10.16420/j.issn.0513-353x.2021-1080

摘要/Abstract

摘要：

‘苏翠1号’梨因其极早熟、汁多味甜、肉质细脆而深受广大消费者喜欢。为了解‘苏翠1号’成熟果实主要代谢产物及代谢通路相关基因表达情况,探究其果实优异性状形成的物质和分子基础,对‘苏翠1号’及其亲本‘翠冠’和‘华酥’成熟果实样本进行代谢组和转录组测序分析。代谢组分析发现,3个品种间,前25个差异代谢产物主要包括有机酸、碳水化合物、氨基酸、甘油磷酸脂、脂肪酸和绿原酸。HPLC测定表明,蔗糖含量在‘苏翠1号’中最高,葡萄糖含量在‘翠冠’中最高,果糖含量在‘苏翠1号’和‘翠冠’中相似,略高于‘华酥’,山梨醇含量在‘苏翠1号’和‘华酥’中均显著低于‘翠冠’;苹果酸含量在‘苏翠1号’中最高,柠檬酸含量在‘华酥’中最高,‘苏翠1号’和‘华酥’总酸含量相近,均高于‘翠冠’。氨基酸含量分析表明,‘苏翠1号’的氨基酸总含量及天冬氨酸、苯丙氨酸、甲硫氨酸含量显著高于‘翠冠’和‘华酥’。转录组GO和KEGG分析表明,相较于‘翠冠’和‘华酥’,‘苏翠1号’有机底物分解过程、脂质代谢、有机酸生物合成、羧酸生物合成、多糖代谢等生物学过程及代谢途径显著富集。进一步挖掘差异基因发现,糖代谢中的SPS、山梨醇代谢中的S6PDH、SDH,TCA循环中的FUM1、ICDH、PDH、OGDHL,氨基酸代谢途径中的GAT、GLSN、LASPO、SAMS,以及脂肪酸代谢中的DGK1、ACCA、KASC2、ADH、PLDA等基因在‘苏翠1号’中显著高表达。这些基因可能与‘苏翠1号’更活跃的蔗糖、山梨醇、氨基酸、甘油磷酯代谢及TCA循环有关。

关键词: 砂梨, 果实品质, 差异代谢产物, 差异基因, qRT-PCR

Abstract:

An early-maturing pear‘Sucui 1’was developed by crossing ‘Huasu’and‘Cuiguan’. The‘Sucui 1’produces fruit with sweet taste,crisp texture and low level of stone cells,so it is well accepted by consumers. To explore biochemical and molecular mechanisms that establish the characteristics of the ripening fruit in‘Sucui 1’,we carried out the metabolic and transcriptomic analysis using UPLC-MS,HPLC,and an amino acid analyzer and RNA-seq approach. The results from the metabolic analysis showed that metabolites such as sugars,organic acids,amino acids and lipids were significantly different between‘Sucui 1’and its parents. Although the total soluble sugar content was similar between‘Sucui 1’and‘Cuiguan’,and the total organic acid content was similar between ‘Sucui 1’and‘Huasu’,the level of sucrose,malate,Asp,Phe,Met,glycerophospholipids and palmitaldehyde was significantly higher in the ripening fruit of‘Sucui 1’. On the other hand,the level of sorbitol and chlorogenic acid was significantly lower in the ripening fruit of‘Sucui 1’compared to ‘Cuiguan’. Differentially expressed genes involved in the biosynthesis pathways of these metabolites were further identified using RNA-Seq. The results from the transcriptomic analysis showed that GO terms such as organic acid biosynthesis,organic substance catabolic process,carbohydrate metabolic process,carboxylic acid biosynthetic process,lipid biosynthetic process and polysaccharide metabolic process were significantly enriched in the comparison of‘Sucui 1’to‘Cuiguan’and‘Huasu’. The higher accumulation of SPS transcripts might be related to the higher level of sucrose in‘Sucui 1’. The higher transcript accumulation of S6PDH and SDH might contribute to its more active sorbitol metabolism. Additionally,the upregulation of FUM1,ICDH,PDH,OGDHL might be related to metabolite accumulations in the TCA cycle. Moreover,differential expression of GAT,GLSN,LASPO,SAMS and DGK1,ACCA,KASC2,ADH2 and PLDA might be related to the higher amino acid and lipid contents in ‘Sucui 1’. These findings provide a foundation for pear quality improvement.

Key words: Pyrus pyrifolia, fruit quality, differentially expressed metabolites, differentially expressed genes, qRT-PCR

中图分类号:

S661.2

王宏, 杨王莉, 蔺经, 杨青松, 李晓刚, 盛宝龙, 常有宏. 早熟砂梨‘苏翠1号’与其亲本‘翠冠’‘华酥’成熟果实差异代谢产物及差异基因比较分析[J]. 园艺学报, 2022, 49(3): 493-508.

WANG Hong, YANG Wangli, LIN Jing, YANG Qingsong, LI Xiaogang, SHENG Baolong, CHANG Youhong. Comparative Metabolic and Transcriptomic Analysis of Ripening Fruit in Pear Cultivars of‘Sucui 1’‘Cuiguan’and‘Huasu’[J]. Acta Horticulturae Sinica, 2022, 49(3): 493-508.

图/表 10

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基因名称 Gene name	基因号 Gene ID	上游引物（5′-3′） Forward primer	下游引物（5′-3′） Reverse Primer
GAPDH	Chr13.g23532	TGGTGTGAACGAGAAGGAAT	CCCTCAACAATCCCAAACC
bZIP53	Chr17.g20327	ACAGGGAGCGGAGCTACAA	AAGAACATGCCGGAGGAGG
ProDH	Chr17.g25295	CTGGTTTACGCTCTCGAGTATG	GAAGAAGGTGGCAGGGATTT
GAT	Chr10.g14827	GACACAGCCATCGACAAAGA	GCCTCTGCATATTCCCAAGTAG
bZIP11	Chr10.g16677	CTCCTGACAAGGGATAACAACC	ACCCATCACCTCCACCAATA
SDH SPS	Chr7.g31955 Chr10.g17334	GTCCGTTCCACTGTATGGTT ACCGGATGGAGATGGGGAT	GCAAAGGAGTGGAGGAGTC CATAATCAATGTAAGGTTAGCAAGCTC

基因名称 Gene name	基因号 Gene ID	上游引物（5′-3′） Forward primer	下游引物（5′-3′） Reverse Primer
GAPDH	Chr13.g23532	TGGTGTGAACGAGAAGGAAT	CCCTCAACAATCCCAAACC
bZIP53	Chr17.g20327	ACAGGGAGCGGAGCTACAA	AAGAACATGCCGGAGGAGG
ProDH	Chr17.g25295	CTGGTTTACGCTCTCGAGTATG	GAAGAAGGTGGCAGGGATTT
GAT	Chr10.g14827	GACACAGCCATCGACAAAGA	GCCTCTGCATATTCCCAAGTAG
bZIP11	Chr10.g16677	CTCCTGACAAGGGATAACAACC	ACCCATCACCTCCACCAATA
SDH SPS	Chr7.g31955 Chr10.g17334	GTCCGTTCCACTGTATGGTT ACCGGATGGAGATGGGGAT	GCAAAGGAGTGGAGGAGTC CATAATCAATGTAAGGTTAGCAAGCTC

分类 Category	代谢产物Compounds	相对含量（Area）/10⁶Relative content
分类 Category	代谢产物Compounds	苏翠1号 Sucui 1	翠冠 Cuiguan	华酥 Huasu
有机酸 Organic acids	甲基丙二酸Methylmalonic acid	2.85 ± 0.66 c	11.73 ± 1.18 a	6.95 ± 0.27 b
	琥珀酸Succinic acid	2.78 ± 0.61 c	11.13 ± 1.10 a	7.11 ± 0.53 b
	柠檬酸Citric acid	3.09 ± 0.09 b	3.00 ± 0.46 b	8.81 ± 0.65 a
	苹果酸L(-)-Malic acid	5.10 ± 0.57 a	2.28 ± 0.02 b	2.62 ± 0.04 b
	丙酸5-Aminolevulinate	3.04 ± 0.21 b	2.04 ± 0.33 c	4.14 ± 0.63 a
	木糖酸D-Xylonic acid	1.86 ± 0.22 b	2.35 ± 0.34 a	1.57 ± 0.28 b
	莽草酸Shikimic acid	1.64 ± 0.08 a	1.47 ± 0.15 a	1.04 ± 0.08 b
	延胡索酸Fumaric acid	0.97 ± 0.07 a	1.04 ± 0.12 a	0.64 ± 0.04 b
碳水化合物	蔗糖D-(+)-Sucrose	2.45 ± 0.25 a	1.77 ± 0.56 c	1.90 ± 0.1 b
Carbohydrates	葡萄糖D-(+)-Glucose	0.50 ± 0.07 b	1.83 ± 0.17 a	0.50 ± 0.13 b
	6-磷酸葡萄糖D-Glucose 6-phosphate	0.68 ± 0.01 c	1.02 ± 0.06 a	0.84 ± 0.05 b
氨基酸Amino acids	天冬氨酸L-Aspartic acid	3.51 ± 0.37 a	3.13 ± 0.26 b	2.02 ± 0.23 c
	DL-正缬氨酸Dl-Norvaline	4.62 ± 0.29 a	3.11 ± 0.28 b	2.76 ± 0.18 c
	苯丙氨酸L-Phenylalanine	2.47 ± 0.29 a	2.21 ± 0.21 a	1.52 ± 0.53 b
	甲硫氨酸L-Methionine	2.07 ± 0.41 a	0.89 ± 0.20 c	1.18 ± 0.23 b
甘油磷脂	溶血磷脂酰乙醇胺16:0 LysoPE 16:0	12.40 ± 0.89 a	9.45 ± 0.56 b	6.08 ± 0.23 c
Glycerophospholipids	溶血磷脂酰胆碱16:0 LysoPC 16:0	10.48 ± 1.32 a	8.87 ± 1.55 b	8.04 ± 0.46 b
	溶血磷脂酰乙醇胺18:2（2n异构）LysoPE 18:2 (2n isomer)	2.24 ± 0.34 a	1.58 ± 0.08 b	1.32 ± 0.09 c
	溶血磷脂酰胆碱18:3（2n异构）LysoPC 18:3 (2n isomer)	2.31 ± 1.07 a	1.16 ± 0.29 b	1.11 ± 0.13 b
	溶血磷脂酰胆碱18:3 LysoPC 18:3	1.53 ± 0.20 a	1.24 ± 0.23 a	1.12 ± 0.04 a
	溶血磷脂酰胆碱18:2 LysoPC 18:2	1.73 ± 0.21 a	1.09 ± 0.13 b	1.25 ± 0.03 b
	溶血磷脂酰胆碱18:1 LysoPC 18:1 (2n isomer)	1.37 ± 0.09 a	1.06 ± 0.40 b	0.78 ± 0.24 c
	溶血磷脂酰胆碱18:2 （2n异构）LysoPC 18:2 (2n isomer)	2.61 ± 0.36 a	0.80 ± 0.15 b	0.82 ± 0.19 b
脂肪酸Fatty acids	棕榈醛Palmitaldehyde	1.07 ± 0.11 a	0.80 ± 0.13 b	0.67 ± 0.13 c
奎宁酸及衍生物Quinate and its derivatives	绿原酸Chlorogenic acid (3-O-Caffeoylquinic acid)	10.21 ± 1.14 b	31.77 ± 3.21 a	34.47 ± 0.57 a

分类 Category	代谢产物Compounds	相对含量（Area）/10⁶Relative content
分类 Category	代谢产物Compounds	苏翠1号 Sucui 1	翠冠 Cuiguan	华酥 Huasu
有机酸 Organic acids	甲基丙二酸Methylmalonic acid	2.85 ± 0.66 c	11.73 ± 1.18 a	6.95 ± 0.27 b
	琥珀酸Succinic acid	2.78 ± 0.61 c	11.13 ± 1.10 a	7.11 ± 0.53 b
	柠檬酸Citric acid	3.09 ± 0.09 b	3.00 ± 0.46 b	8.81 ± 0.65 a
	苹果酸L(-)-Malic acid	5.10 ± 0.57 a	2.28 ± 0.02 b	2.62 ± 0.04 b
	丙酸5-Aminolevulinate	3.04 ± 0.21 b	2.04 ± 0.33 c	4.14 ± 0.63 a
	木糖酸D-Xylonic acid	1.86 ± 0.22 b	2.35 ± 0.34 a	1.57 ± 0.28 b
	莽草酸Shikimic acid	1.64 ± 0.08 a	1.47 ± 0.15 a	1.04 ± 0.08 b
	延胡索酸Fumaric acid	0.97 ± 0.07 a	1.04 ± 0.12 a	0.64 ± 0.04 b
碳水化合物	蔗糖D-(+)-Sucrose	2.45 ± 0.25 a	1.77 ± 0.56 c	1.90 ± 0.1 b
Carbohydrates	葡萄糖D-(+)-Glucose	0.50 ± 0.07 b	1.83 ± 0.17 a	0.50 ± 0.13 b
	6-磷酸葡萄糖D-Glucose 6-phosphate	0.68 ± 0.01 c	1.02 ± 0.06 a	0.84 ± 0.05 b
氨基酸Amino acids	天冬氨酸L-Aspartic acid	3.51 ± 0.37 a	3.13 ± 0.26 b	2.02 ± 0.23 c
	DL-正缬氨酸Dl-Norvaline	4.62 ± 0.29 a	3.11 ± 0.28 b	2.76 ± 0.18 c
	苯丙氨酸L-Phenylalanine	2.47 ± 0.29 a	2.21 ± 0.21 a	1.52 ± 0.53 b
	甲硫氨酸L-Methionine	2.07 ± 0.41 a	0.89 ± 0.20 c	1.18 ± 0.23 b
甘油磷脂	溶血磷脂酰乙醇胺16:0 LysoPE 16:0	12.40 ± 0.89 a	9.45 ± 0.56 b	6.08 ± 0.23 c
Glycerophospholipids	溶血磷脂酰胆碱16:0 LysoPC 16:0	10.48 ± 1.32 a	8.87 ± 1.55 b	8.04 ± 0.46 b
	溶血磷脂酰乙醇胺18:2（2n异构）LysoPE 18:2 (2n isomer)	2.24 ± 0.34 a	1.58 ± 0.08 b	1.32 ± 0.09 c
	溶血磷脂酰胆碱18:3（2n异构）LysoPC 18:3 (2n isomer)	2.31 ± 1.07 a	1.16 ± 0.29 b	1.11 ± 0.13 b
	溶血磷脂酰胆碱18:3 LysoPC 18:3	1.53 ± 0.20 a	1.24 ± 0.23 a	1.12 ± 0.04 a
	溶血磷脂酰胆碱18:2 LysoPC 18:2	1.73 ± 0.21 a	1.09 ± 0.13 b	1.25 ± 0.03 b
	溶血磷脂酰胆碱18:1 LysoPC 18:1 (2n isomer)	1.37 ± 0.09 a	1.06 ± 0.40 b	0.78 ± 0.24 c
	溶血磷脂酰胆碱18:2 （2n异构）LysoPC 18:2 (2n isomer)	2.61 ± 0.36 a	0.80 ± 0.15 b	0.82 ± 0.19 b
脂肪酸Fatty acids	棕榈醛Palmitaldehyde	1.07 ± 0.11 a	0.80 ± 0.13 b	0.67 ± 0.13 c
奎宁酸及衍生物Quinate and its derivatives	绿原酸Chlorogenic acid (3-O-Caffeoylquinic acid)	10.21 ± 1.14 b	31.77 ± 3.21 a	34.47 ± 0.57 a

氨基酸Amino acids	苏翠1号Sucui 1	翠冠Cuiguan	华酥Huasu
L-天冬氨酸 Asp	351.00 ± 37.02 a	313.00 ± 26.38 b	202.00 ± 23.02 c
L-苯丙氨酸 Phe	247.00 ± 28.92 a	221.00 ± 21.22 b	152.00 ± 53.21 c
L-甲硫氨酸 Met	207.00 ± 40.93 a	100.70 ± 19.96 b	118.00 ± 21.82 b
L-(+)-赖氨酸 Lys	102.00 ± 15.51 a	46.90 ± 12.10 b	111.00 ± 10.05 a
L-谷氨酸 Glu	58.80 ± 9.20 a	51.40 ± 23.28 a	44.5 ± 12.37 b
L-(-)-苏氨酸 Thr	41.80 ± 6.19 a	38.70 ± 3.64 b	26.00 ± 2.29 c
L-丙氨酸 Ala	41.40 ± 5.83 a	28.50 ± 2.29 b	24.50 ± 3.67 b
L-(+)-精氨酸 Arg	23.20 ± 4.68 b	31.40 ± 4.06 a	18.30 ± 8.30 c
L-(-)-酪氨酸 Tyr	20.70 ± 2.95 a	22.40 ± 3.12 a	6.16 ± 0.27 b
L-半胱氨酸 Cys	9.12 ± 1.47 b	14.20 ± 0.87 a	5.14 ± 0.92 c
L-异亮氨酸 Ile	4.19 ± 0.17 a	3.04 ± 0.45 b	4.91 ± 0.79 a
L-组氨酸 His	3.61 ± 0.78 a	0.75 ± 0.17 c	2.92 ± 0.12 b
L-亮氨酸 Leu	3.55 ± 0.40 b	2.46 ± 0.31 c	4.08 ± 0.12 a
L-缬氨酸 Val	52.10 ± 0.02 a	54.50 ± 0.01 a	40.42 ± 0.01 b
L-脯氨酸 Pro	8.69 ± 0.02 c	15.12 ± 0.65 a	13.20 ± 0.68 b
总量 Total content	1 114.47	966.35	772.73