波罗蜜柠檬酸合酶基因(<i>CS</i>)的表达及柠檬酸积累相关性分析

李思彤; 李真琴; 林婉彤; 王俊宁

波罗蜜柠檬酸合酶基因(CS)的表达及柠檬酸积累相关性分析

1.
广东海洋大学滨海农业学院，广东　湛江，524088

基金项目: 农科教合作人才培养基地资助项目(GDOU2013040301)；教育部卓越农林人才培养计划资助项目(园艺, GDOU2014041204)；广东省园艺和园林专业实践技能型农林人才培养模式改革试点资助项目(GDOU2014041208)共同资助

详细信息

作者简介:
李思彤（1999 —），女，硕士研究生，研究方向：采后生理与分子生物学。 E-mail：lisitong@163.com

通讯作者:
王俊宁（1978 —），女，博士，教授，研究方向：果蔬采后生理与分子生物学. E-mail：wangjunningb@126.com

中图分类号: S667.8
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出版历程
- 收稿日期: 2023-10-11
- 修回日期: 2023-12-23
- 网络出版日期: 2024-03-28

Expression and Correlation Analysis of Citrate Synthase Gene (CS) in Pomelo and Citric Acid Accumulation

1.
Coastal Agricultural College of Guangdong Ocean University , Zhanjiang , Guangdong　524088, China

摘要

摘要: 目的有机酸是果实的重要风味物质之一，而柠檬酸合酶 (CS)是果实有机酸代谢过程中重要的限速酶之一。克隆波罗蜜柠檬酸合酶基因AheCS，测定波罗蜜果实采后不同处理下的柠檬酸含量，分析AheCS基因的生物学功能，研究柠檬酸含量与AheCS基因相对表达量的相关性，探究AheCS基因在波罗蜜果实有机酸代谢中的可能作用。方法以海大2号波罗蜜果实为材料，采用0.5 mg·L⁻¹ 1-MCP和1000 mg·L⁻¹ ETH (40%)处理，研究室温(22±1) ℃、相对湿度90% 条件下波罗蜜果实成熟过程中柠檬酸的动态变化，克隆获得3个波罗蜜的CS基因(AheCS1、AheCS2和AheCS3)，对其生物信息学进行了分析，同时分析了AheCS1/2/3在不同采后处理下(自然成熟、外源乙烯催熟和1-MCP延缓成熟)的相对表达情况及其与柠檬酸含量的相关性。结果随着波罗蜜果实的自然成熟，果实中柠檬酸含量呈先上升后下降的变化趋势；外源ETH处理加速了柠檬酸的合成速率，提前了峰值出现的时间；1-MCP处理抑制了贮藏前期柠檬酸含量下降速率，推迟了高峰出现时间。生物信息学分析发现，AheCS1/2/3基因的开放阅读框(ORF)长为1422～1827bp。AheCS1/2/3蛋白都含有柠檬酸合酶保守结构域‘WPNVDAHS’，属于柠檬酸合酶家族成员。AheCS1/2/3氨基酸序列分别与柑橘CsCS(MH_048698.1) 、川桑MnCs(XP010087965.1) 、红掌AaCS(JAT55223.1)亲缘关系最近，相似性分别达到86.49%、97%、86%。qRT-PCR结果显示： AheCS1/2/3基因在果实自然成熟(CK)前期表达量低，而后期表达量高；外源ETH处理提前了AheCS 1的表达，整体提高了AheCS 2/3的表达量；而1-MCP处理推迟了AheCS1/2/3表达峰值出现时间，但增加了AheCS1/2/3成熟后期的表达量。相关性分析发现，波罗蜜果实成熟过程中柠檬酸含量与AheCS1/2/3基因表达呈一定相关性，其中AheCS2相关性较为显著。结论 AheCS2是参与调控波罗蜜成熟过程中柠檬酸积累的潜在基因，这为进一步研究波罗蜜AheCS基因的功能及遗传改良等提供理论依据。
- 波罗蜜 /
- 柠檬酸 /
- AheCS基因 /
- 克隆 /
- 基因表达
Abstract: : Objective Rganic acids are one of the important flavor substances in fruits, Citrate synthase (CS) plays a pivotal role as a rate-limiting enzyme in the organic acid metabolism of fruits. The citric acid synthase gene AheCS was cloned, and the citric acid content of jackfruit postharvest under different treatments was determined. The biological function of AheCS gene and the correlation between citric acid content and relative expression of AheCS gene were analyzed, and the possible role of AheCS gene in the metabolism of organic acids in jackfruit was discussed. Method Haida 2 jackfruit were utilized, treated with 0.5 mg·L⁻¹ 1-MCP and 1000 mg·L⁻¹ ETH (40%), and subjected to investigation into the dynamic changes in citric acid during jackfruit fruit ripening under room temperature (22±1°C) and 90% RH conditions. Three jackfruit CS genes (AheCS1, AheCS2, and AheCS3) were cloned, and their bioinformatics were analyzed. Its bioinformatics was analyzed, as well as AheCS1/2/3 under different post-harvest treatments (natural ripening, exogenous ethylene ripening, and 1-MCP delayed ripening). Result As jackfruit underwent natural ripening, citric acid content exhibited an initial increase followed by a decline. Exogenous ETH treatment accelerated the synthesis rate of citric acid, advancing the time of peak appearance. 1-MCP treatment inhibited the rate of citric acid decline in the early storage period, thus delaying the peak. Bioinformatics analysis revealed that the open reading frames (ORFs) of AheCS1/2/3 genes were 1422 bp to 1827 bp long. AheCS1/2/3 proteins contained the conserved 'WPNVDAHS' domain of citrate synthase, classifying them as members of the citrate synthase family. The amino acid sequences of AheCS1/2/3 showed the closest phylogenetic relationships with citrus CsCS (MH_048698.1), mulberry MnCs (XP010087965.1), and Anthurium AaCS (JAT55223.1), with similarities reaching 86.49%, 97%, and 86%, respectively. qRT-PCR results indicated that the expression of AheCS1/2/3 genes was low in the early stage of natural fruit ripening (CK) and increased in the later stage. Exogenous ETH treatment advanced the expression of AheCS1 and overall increased the expression of AheCS2/3. 1-MCP treatment delayed the increase in the expression of Peak occurrence time of AheCS1/2/3 but increased their expression in the later stages of maturity. Correlation analysis revealed a positive correlation between citric acid content and the expression of AheCS1/2/3 genes during jackfruit fruit ripening, with AheCS2 reaching a significant level. Conclusion The accumulation of citric acid during jackfruit maturation may be regulated by the AheCS2 genes, providing a foundation for further research on the functionality and genetic improvement of jackfruit AheCS genes.
- jackfruit /
- citrate acid /
- AheCS genes /
- cloning /
- gene expression

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图 1 不同处理对柠檬酸含量的影响

Figure 1. Effect of different treatments on the contents of citric acid

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图 2 AheCS1, AheCS2, AheCS3基因的克隆

M: DL2000 DNA Marker；CS1/2/3: 波罗蜜目的基因

Figure 2. Cloning of AheCS1, AheCS2, AheCS3 genes

M: DL2000 DNA Marker；CS1/2/3: Jackfruit genes

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图 3 AheCS1/2/3编码蛋白的三级结构预测

Figure 3. Prediction of the tertiary structure of the protein encoded by the AheCS1/2/3

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图 4 AheCS1/2/3 蛋白的多重序列比对

Co：油菜；Jc：麻风树；Mn：川桑；Cm：柚；Pp：桃；Ps：西伯利亚杏；Mb：山荆子；Cu：葫芦；Aa：红掌；Pm：梅；Pas：牡丹；Ahe：波罗蜜；Cs：柑橘；Gu：甘草；AT：拟南芥.

Figure 4. Alignment of the amino acid sequence of AheCS1/2/3protein

Co: Camellia oleifera Abel.; Jc: Jatropha curcas L.; Mn: Morus notabilis Schneid.; Cm; Citrus maxima (Burm) Merr.; Pp: Prunus persica; Ps: Prunus sibirica L.; Mb: Malus baccata (L.) Borkh.; Cu: Cucurbita cv.; Aa: Anthurium andraeanum.; Pm：Armeniaca mume Sieb.; Ps: Paeonia Suffruticosa.; Ahe：Artocarpus heterophyllus Lam.; Cs: Citrus reticulata Blanco.; Gu: Glycyrrhiza uralensis Fisch.; AT: Arabidopsis thaliana.

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图 5 AheCS1/2/3 蛋白保守基序分析

Figure 5. AheCS1/2/3 protein conserved motif analysis

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图 6 不同物种CS基因的系统发育树分析

St：马铃薯；Cf：甜椒；Cc：中粒咖啡；Co：油菜；As：山杏；Ps：牡丹；Js：泡核桃；Pp：桃；PpN：砂梨：Mb：山荆子；Pc：豆梨；AT：拟南芥；Os：水稻；Sb：高粱；Ahe：波罗蜜；SL：番茄

Figure 6. Phylogenetic tree analysis of CS in different species

St: Solanum tuberosum L.; Cf: Capsicum frutescens L. ; Cc: Coffea canephora Pierre ex Froehn.; Co: Camellia oleifera; As: Armeniaca sibirica (L.) Lam.; Ps: PaeoniaSuffruticosa; Js: Juglans sigillata Dode; Pp: Prunus persica; PpN: Pyrus pyrifolia (Burm. f.) Nakai; Mb: Malus baccata (L.) Borkh.; Pc: Pyrus calleryana Decne.; AT: Arabidopsis thaliana (L.) Heynh.; Os: Oryza sativa L.; Sb: Sorghum 'Bicolor'(L.) Moench; Ahe: Artocarpus heterophyllus Lam. SL: Solanum lycopersicum L.

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图 7 成熟过程中AheCS1、AheCS2和AheCS3基因的表达分析

Figure 7. Relative expression levels of AheCS1/2/3 genes

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表 1 AheCS1、AheCS2和AheCS3基因全长引物序列

Table 1. Primer sequences of AheCS1, AheCS2 and AheCS3 genes

引物名称 Primer names	核苷酸序列(5′-3′) Nucleic acid sequences
AheCS1-for AheCS1-rev AheCS2-for AheCS2-rev AheCS3-for AheCS3-rev	5′-ATGGCCACCGGACAGCTATTCTCGCG-3′ 5′-TCACTTGGTGTAAAGAACGTCCTCCCATG-3′ 5′-ATGGTGTTCTTCAGGGGCGTGTCTGTGC-3′ 5′-TCAAGACGAAGCCGCTTTCTTGCAGTAA-3′ 5′-ATGGAATTGCCAGTCACGGCACGAGC-3′ 5′-TTAAATGCCAGAACCCGCCAGCCGG-3′

下载: 导出CSV

表 2 基因荧光定量引物序列

Table 2. Gene Quantitative PCR primer sequence

引物名称	核苷酸序列(5′-3′)
Primer names	Nucleic acid sequences
AheCS1-qup	5′-AGAATCAAGCACTAAGGGACG-3′
AheCS1-qdw	5′-TTCAGGAATTTGTGGAGGC-3′
AheCS2-qup	5′-GCCTCCCATCCTAACAGAAA-3′
AheCS2-qdw	5′-CGCTCGGTCCCATACTAACT-3′
AheCS3-qup	5′- CCAACCGAGTTCTTCCCTG-3′
AheCS3-qdw	5′- GATAATGCCGCAACCAAAC -3′
AheGAPDH-qup	5′- TTGAAGGGTGGNGCNAARAARG -3′
AheGAPDH-qdw	5′- ATAACCCCAYTCRTTRTCRTAC -3′
AheGAPDH 为内参基因。 AheGAPDH is internal reference gene.

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表 3 AheCS1/2/3 蛋白的理化性质

Table 3. Physical and chemical properties of AheCS1/2/3 protein

基因名称 Gene name	氨基酸数 Number of amino acids	分子质量 /kD mole cular weight	等电点 The- oretical pI	脂肪系数 Aliphatic index	不稳定系数 Instability index	亲水性 Grand average of hydropathicity	含量最多的 3 种氨基酸 3 Most abundant amino acids
AheCS1	608	151.18	4.96(酸性)	25.29	45.73(不稳定)	0.698	Ala 25.3% Gly25.7% Thr27.5%
AheCS2	473	52.89	7.20(碱性)	26.37	40.34(不稳定)	0.652	Gly24.6% Ala26.4% Thr29.7%
AheCS3	513	56.42	8.15(碱性)	25.02	47.61(不稳定)	0.748	Ala25.0% Gly25.4% Thr26.3%

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表 5 AheCS1/2/3蛋白磷酸化和糖基化位点及二级结构组分

Table 5. Phosphorylation and glycosylation sites and secondary structure components of AheCS1/2/3 protein

基因名称 Gene name	糖基化位点数/个 Number of glycosy	磷酸化位点数/个 number of phosphorylation sites/piece			二级结构组分/% secondary structure component
基因名称 Gene name	糖基化位点数/个 Number of glycosy	丝氨酸 Ser	苏氨酸 Thr	酪氨酸 Tyr	α-螺旋 α-helix	延长链 Extend chain	β-转角 β-turn	无规则卷曲 random coil
AheCS1	1	59	55	24	40.95%	16.78%	9.54%	32.73%
AheCS2	0	17	10	9	53.70%	8.88%	7.82%	29.60%
AheCS3	0	28	9	11	47.85%	9.96%	5.08%	37.11%

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表 4 AheCS1/2/3 蛋白的亚细胞定位预测

Table 4. Prediction of subcellular localization of AheCS1/2/3 protein

定位 Location	AheCS1	AheCS2	AheCS3
细胞核 Nuclear	0.04	0.03	0.00
质膜 Plasma membrane	0.02	0.12	0.00
胞外 Extra-cellular	0.00	0.00	0.00
细胞质Cytoplasmic	8.81	0.00	0.00
线粒体 Mitochondria	0.07	6.94	0.00
细胞质内层 Endoplasm retic	0.08	0.00	0.33
过氧化物酶体Peroxisomal	0.00	5.91	9.41
高尔基体Golgi	0.00	0.01	0.14
叶绿体 Chloroplast	0.66	0.00	0.12
液泡 Vacuolar	0.31	0.00	0.00

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表 6 有机酸与AheCS1/2/3 基因之间相关性分析

Table 6. Correlation analysis between organic acids and AheCS1/2/3 genes

指标	柠檬酸	AheCS1	AheCS2	AheCS3
柠檬酸	1
AheCS1	0.450	1
AheCS2	0.887*	0.088	1
AheCS3	0.766	0.339	0.521	1
* 在 0.05 级别（双尾），相关性显著。 At the 0.05 level (two-tailed), the correlation is significant*.

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参考文献(42)

[1]	LI J X, ZHANG C L, LIU H, et al. Profiles of sugar and organic acid of fruit juices: A comparative study and implication for authentication [J]. Journal of Food Quality, 2020, 2020: 7236534.
[2]	马晓倩. 柠檬酸含量对冰糖橙果实贮藏性能的影响及其代谢相关基因分析 [D]. 长沙: 湖南农业大学, 2020. MA X Q. Effect of citric acid content on storage performance of 'Bingtang' Sweet Orange and expression analysis of genes in citrate metabolism [D]. ChangSha: Hunan Agricultural University, 2020. (in Chinese)
[3]	SADKA A, DAHAN E, COHEN L, et al. Aconitase activity and expression during the development of lemon fruit [J]. Physiologia Plantarum, 2000, 108(3): 255−262. doi: 10.1034/j.1399-3054.2000.108003255.x
[4]	BLANKE M M, LENZ F. Fruit photosynthesis[J]. Plant, Cell & Environment, 1989, 12(1): 31-46.
[5]	KOYAMA H, TAKITA E, KAWAMURA A, et al. Over expression of mitochondrial citrate synthase gene improves the growth of carrot cells in Al-phosphate medium [J]. Plant and Cell Physiology, 1999, 40(5): 482−488. doi: 10.1093/oxfordjournals.pcp.a029568
[6]	PRACHAROENWATTANA I, CORNAH J E, SMITH S M. Arabidopsis peroxisomal citrate synthase is required for fatty acid respiration and seed germination [J]. The Plant Cell, 2005, 17(7): 2037−2048. doi: 10.1105/tpc.105.031856
[7]	OHLROGGE J, POLLARD M, BAO X, et al. Fatty acid synthesis: From CO₂ to functional genomics [J]. Biochemical Society Transactions, 2000, 28(6): 567−573. doi: 10.1042/bst0280567
[8]	SCHNARRENBERGER C, MARTIN W. Evolution of the enzymes of the citric acid cycle and the glyoxylate cycle of higher plants. A case study of endosymbiotic gene transfer [J]. European Journal of Biochemistry, 2002, 269(3): 868−883. doi: 10.1046/j.0014-2956.2001.02722.x
[9]	ZHAO H N, CHEN G J, SANG L N, et al. Mitochondrial citrate synthase plays important roles in anthocyanin synthesis in petunia [J]. Plant Science:an International Journal of Experimental Plant Biology, 2021, 305: 110835.
[10]	TOVAR-MÉNDEZ A, MIERNYK J A, RANDALL D D. Regulation of pyruvate dehydrogenase complex activity in plant cells [J]. European Journal of Biochemistry, 2003, 270(6): 1043−1049. doi: 10.1046/j.1432-1033.2003.03469.x
[11]	SADKA A, DAHAN E, OR E, et al. Comparative analysis of mitochondrial citrate synthase gene structure, transcript level and enzymatic activity in acidless and acid-containing Citrus varieties [J]. Functional Plant Biology, 2001, 28(5): 383. doi: 10.1071/PP00136
[12]	CANEL C, BAILEY-SERRES J N, ROOSE M L. Molecular characterization of the mitochondrial citrate synthase gene of an acidless pummelo ( Citrus maxima) [J]. Plant Molecular Biology, 1996, 31(1): 143−147. doi: 10.1007/BF00020613
[13]	IANNETTA P P M, ESCOBAR N M, ROSS H A, et al. Identification, cloning and expression analysis of strawberry ( Fragaria x ananassa) mitochondrial citrate synthase and mitochondrial malate dehydrogenase [J]. Physiologia Plantarum, 2004, 121(1): 15−26. doi: 10.1111/j.0031-9317.2004.00302.x
[14]	叶思诚, 姚小华, 王开良, 等. 油茶柠檬酸合成酶(CS)基因的克隆和表达分析 [J]. 植物研究, 2016, 36(4):556−564. doi: 10.7525/j.issn.1673-5102.2016.04.011 YE S C, YAO X H, WANG K L, et al. Cloning and expression analysis of citrate synthase(CS)gene in Camellia oleifera [J]. Bulletin of Botanical Research, 2016, 36(4): 556−564. (in Chinese) doi: 10.7525/j.issn.1673-5102.2016.04.011
[15]	王滕旭, 李正国, 杨迎伍, 等. 甜橙柠檬酸合酶基因的克隆及其表达分析 [J]. 中国农学通报, 2010, 26(10):65−69. WANG T X, LI Z G, YANG Y W, et al. Cloning and expression analysis of citrate synthase gene in orange [J]. Chinese Agricultural Science Bulletin, 2010, 26(10): 65−69. (in Chinese)
[16]	赵宝庆, 任伟超, 王震, 等. 酸橙柠檬酸合酶基因电子克隆和生物信息学分析 [J]. 东北林业大学学报, 2019, 47(12):79−83. ZHAO B Q, REN W C, WANG Z, et al. In silico cloning and bioinformatics analysis of citrate synthase gene from Citrus aurantium [J]. Journal of Northeast Forestry University, 2019, 47(12): 79−83. (in Chinese)
[17]	童晋, 詹高淼, 王新发, 等. 油菜柠檬酸合酶基因的克隆及在逆境下的表达 [J]. 作物学报, 2009, 35(1):33−40. doi: 10.3724/SP.J.1006.2009.00033 TONG J, ZHAN G M, WANG X F, et al. Cloning of citrate synthase gene in rapeseed ( Brassica napus L. ) and its expression under stresses [J]. Acta Agronomica Sinica, 2009, 35(1): 33−40. (in Chinese) doi: 10.3724/SP.J.1006.2009.00033
[18]	王俊宁, 刘建文, 李凤娣, 等. 菠萝蜜果实采后糖、酸、蛋白质和V_C含量的变化 [J]. 西南师范大学学报(自然科学版), 2015, 40(3):80−85. WANG J N, LIU J W, LI F D, et al. On changes in starch, sugar, acids, VC and proteins contents of jackfruit during ripening process [J]. Journal of Southwest China Normal University (Natural Science Edition), 2015, 40(3): 80−85. (in Chinese)
[19]	夏春华, 杨转英, 叶春海, 等. 不同株系菠萝蜜果肉中可溶性糖和有机酸的分析 [J]. 果树学报, 2014, 31(4):648−652. XIA C H, YANG Z Y, YE C H, et al. Analysis of contents and compositions of soluble sugars and organic acids in different jackfruit( Artocarpus heterophyllus) lines [J]. Journal of Fruit Science, 2014, 31(4): 648−652. (in Chinese)
[20]	王俊宁, 吕庆芳, 丰锋, 等. 湿包类型菠萝蜜采后呼吸跃变和主要成分的变化 [J]. 园艺学报, 2014, 41(2):329−334. doi: 10.3969/j.issn.0513-353X.2014.02.014 WANG J N, LYU Q F, FENG F, et al. Changes in respiration, ethylene and biochemical values of soft jackfruits during the ripening period [J]. Acta Horticulturae Sinica, 2014, 41(2): 329−334. (in Chinese) doi: 10.3969/j.issn.0513-353X.2014.02.014
[21]	王俊宁, 陈文耀, 丰锋, 等. 乙烯利处理对菠萝蜜果实催熟的影响 [J]. 广东农业科学, 2014, 41(3):94−98. doi: 10.3969/j.issn.1004-874X.2014.03.023 WANG J N, CHEN W Y, FENG F, et al. Effect of ethephon treatment on physiological index of jackfruit after harvest [J]. Guangdong Agricultural Sciences, 2014, 41(3): 94−98. (in Chinese) doi: 10.3969/j.issn.1004-874X.2014.03.023
[22]	王俊宁, 陈俊鹏, 弓德强, 等. 1-MCP处理对菠萝蜜采后生理效应的影响 [J]. 江西农业大学学报, 2014, 36(1):56−61,96. doi: 10.3969/j.issn.1000-2286.2014.01.009 WANG J N, CHEN J P, GONG D Q, et al. Effects of 1-MCP treatment on post-harvest physiology of jackfruit( Artocarpus heterophyllus lam. ) [J]. Acta Agriculturae Universitatis Jiangxiensis, 2014, 36(1): 56−61,96. (in Chinese) doi: 10.3969/j.issn.1000-2286.2014.01.009
[23]	GAO Y Y, YAO Y L, CHEN X, et al. Metabolomic and transcriptomic analyses reveal the mechanism of sweet-acidic taste formation during pineapple fruit development [J]. Frontiers in Plant Science, 2022, 13: 971506. doi: 10.3389/fpls.2022.971506
[24]	ZHANG X X, WEI X X, ALI M M, et al. Changes in the content of organic acids and expression analysis of citric acid accumulation-related genes during fruit development of yellow ( Passiflora edulis f. flavicarpa) and purple ( Passiflora edulis f. edulis) passion fruits [J]. International Journal of Molecular Sciences, 2021, 22(11): 5765. doi: 10.3390/ijms22115765
[25]	李雪梅. 砂梨果实有机酸含量及代谢相关酶活性动态变化研究[D]. 武汉: 华中农业大学, 2008. LI X M. Studies on dynamic changes of organic acid contents and metabolizing enzyme activities in sand pear fruit[D]. Wuhan: Huazhong Agricultural University, 2008. (in Chinese)
[26]	朱磊, 陈芸华, 胡禧熙, 等. 葡萄有机酸的研究进展 [J]. 中外葡萄与葡萄酒, 2022, (6):88−95. ZHU L, CHEN Y H, HU X X, et al. Research progress of organic acids in grape [J]. Sino-Overseas Grapevine & Wine, 2022(6): 88−95. (in Chinese)
[27]	杨滢滢. ‘纽荷尔’脐橙果实柠檬酸合成与降解机理的研究[D]. 南昌: 江西农业大学, 2017. YANG Y Y. Research on synthesis and degradation of citric acid in’Newhall’ navel orange fruit[D]. Nanchang: Jiangxi Agricultural University, 2017. (in Chinese)
[28]	张秀梅, 杜丽清, 孙光明, 等. 菠萝果实发育过程中有机酸含量及相关代谢酶活性的变化 [J]. 果树学报, 2007, 24(3):381−384. doi: 10.3969/j.issn.1009-9980.2007.03.025 ZHANG X M, DU L Q, SUN G M, et al. Changes in organic acid concentrations and the relative enzyme activities during the development of Cayenne pineapple fruit [J]. Journal of Fruit Science, 2007, 24(3): 381−384. (in Chinese) doi: 10.3969/j.issn.1009-9980.2007.03.025
[29]	文涛, 熊庆娥, 曾伟光, 等. 脐橙果实发育中有机酸合成代谢酶活性变化的研究 [J]. 四川农业大学学报, 2001, 19(1):27−30. WEN T, XIONG Q E, ZENG W G, et al. Study on the change of organic acid synthetase activity during fruit development of navel orange ( Citrus sinesis osbeck) [J]. Journal of Sichuan Agricultural University, 2001, 19(1): 27−30. (in Chinese)
[30]	张沛宇. 采后乙烯利脱绿对柠檬果实色泽、风味及抗氧化能力的影响研究[D]. 重庆: 西南大学, 2019. ZHANG P Y. Effects of postharvest ethephon degreening on the color, flavor quality and antioxidant capacity of lemon (Citrus limon (L. ) burm. f. ) fruit[D]. Chongqing: Southwest University, 2019. (in Chinese)
[31]	LIM S, LEE J G, LEE E J. Comparison of fruit quality and GC-MS-based metabolite profiling of kiwifruit 'Jecy green': Natural and exogenous ethylene-induced ripening [J]. Food Chemistry, 2017, 234: 81−92. doi: 10.1016/j.foodchem.2017.04.163
[32]	郭香凤, 向进乐, 史国安, 等. 1-MCP对凯特杏果实采后糖酸组分与含量的影响 [J]. 保鲜与加工, 2008, 8(5):30−33. GUO X F, XIANG J Y, SHI G A, et al. Effects of 1-methylcydopropene on sugar and organic acid constituents in katy apricot [J]. Storage and Process, 2008, 8(5): 30−33. (in Chinese)
[33]	弓德强, 李敏, 高兆银, 等. 1-甲基环丙烯处理对樱桃番茄果实低温贮藏品质的影响 [J]. 食品与发酵工业, 2022, 48(4):116−122,129. GONG D Q, LI M, GAO Z Y, et al. Effect of 1-methylcyclopropene treatment on quality of cherry tomatoes stored at low temperature [J]. Food and Fermentation Industries, 2022, 48(4): 116−122,129. (in Chinese)
[34]	石岩. 小金海棠柠檬酸合成酶基因MxCS₂的克隆与功能分析[D]. 哈尔滨: 东北农业大学, 2016. SHI Y. Cloning and functional analysis of Malus xiaojinensis citrate synthase gene MxCS₂[D]. Harbin: Northeast Agricultural University, 2016. (in Chinese)
[35]	张小红. 琯溪蜜柚(Citrus grandis(L. )osbeck. cv. guanxi Miyou)果实采后酸代谢研究[D]. 福州: 福建农林大学, 2005. ZHANG X H. Studies on the metabolism of organic acids in Citrus guanxi-miyou (Citrus grandis (L. ) osbeck. cv. guanxi Miyou) fruit after harvest[D]. Fuzhou: Fujian Agriculture and Forestry University, 2005. (in Chinese)
[36]	张春梅. 枣糖酸代谢及其驯化的分子机制研究[D]. 杨凌: 西北农林科技大学, 2016. ZHANG C M. Molecular mechanism related to the metabolism of sugar, acid and domestication for Ziziphus jujuba mill[D]. Yangling: Northwest A & F University, 2016. (in Chinese)
[37]	ZHANG G F, XIE S X. Influence of water stress on the citric acid metabolism related gene expression in the ponkan fruits [J]. Agricultural Sciences, 2014, 5(14): 1513−1521. doi: 10.4236/as.2014.514162
[38]	文涛, 熊庆娥, 曾伟光, 等. 脐橙果实发育过程中有机酸合成代谢酶活性的变化 [J]. 园艺学报, 2001, 28(2):161−163. doi: 10.3321/j.issn:0513-353X.2001.02.015 WEN T, XIONG Q E, ZENG W G, et al. Changes of organic acid synthetase activity during fruit development of navel orange( Citrus sinesis osbeck) [J]. Acta Horticulturae Sinica, 2001, 28(2): 161−163. (in Chinese) doi: 10.3321/j.issn:0513-353X.2001.02.015
[39]	李航. 两种中国樱桃果实糖酸积累和代谢相关酶及其基因表达的研究[D]. 雅安: 四川农业大学, 2019. LI H. Study on sugar and acid accumulation, metabolism enzymes activities and gene expression of two Chinese cherry fruits[D]. Yaan: Sichuan Agricultural University, 2019. (in Chinese)
[40]	翁金洋. 梅和杏果实糖酸变化规律及有机酸代谢差异研究[D]. 南京: 南京农业大学, 2017. WENG J Y. Studies on the change regulation of sugar and acid and the metabolism of organic acids in Prunus mume and apricot fruits[D]. Nanjing: Nanjing Agricultural University, 2017. (in Chinese)
[41]	王羊, 邓倩, 邓群仙, 等. “蜀脆枣” 果实发育过程中糖酸代谢相关基因表达分析 [J]. 西北农业学报, 2022, 31(7):876−885. doi: 10.7606/j.issn.1004-1389.2022.07.009 WANG Y, DENG Q, DENG Q X, et al. Analysis of sugar and organic acid metabolism-related genes expression during development of Ziziphus jujuba cv. “Shucuizao” fruits [J]. Acta Agriculturae Boreali-occidentalis Sinica, 2022, 31(7): 876−885. (in Chinese) doi: 10.7606/j.issn.1004-1389.2022.07.009
[42]	LIU J H, CHI G H, JIA C H, et al. Function of a citrate synthase gene (MaGCS) during postharvest banana fruit ripening [J]. Postharvest Biology and Technology, 2013, 84: 43−50. doi: 10.1016/j.postharvbio.2013.04.005