波罗蜜柠檬酸合酶基因（<i>AheCS</i>）的表达及其与柠檬酸积累的相关性分析

李思彤; 李真琴; 林婉彤; 王俊宁

doi:10.19303/j.issn.1008-0384.2024.02.005

波罗蜜柠檬酸合酶基因（AheCS）的表达及其与柠檬酸积累的相关性分析

doi: 10.19303/j.issn.1008-0384.2024.02.005

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项目（GDOU2013040301）；教育部卓越农林人才培养计划项目（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项目（GDOU2014041208）

详细信息

作者简介:
李思彤（1999 —），女，硕士研究生，主要从事采后生理与分子生物学研究， E-mail：lisitong0228@163.com

通讯作者:
王俊宁（1978 —），女，博士，教授，主要从事果蔬采后生理与分子生物学研究，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
- 刊出日期: 2024-02-28

Expression and Function of Citrate Synthase Gene in Jackfruit

Coastal Agricultural College, Guangdong Ocean University, Zhanjiang, Guangdong　524088, China

摘要

摘要: 目的分析波罗蜜柠檬酸合酶基因（Artocarpus heterophyllus citrate synthase，AheCS）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，为探究AheCS基因在波罗蜜（Artocarpus heterophyllus Lam. , Ahe）果实有机酸代谢中的可能作用提供参考。方法以海大2号波罗蜜果实为材料，采用0.5 mg·L⁻¹ 1-甲基环丙烯（1-Methylcyclopropene,1-MCP）和1000 mg·L⁻¹ 外源乙烯利（ethrel , 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），对其进行生物信息学分析，同时分析3个AheCS基因在不同采后处理下（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1422～1827 bp，编码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蛋白氨基酸序列分别与柑橘（Citrus reticulata Blanco.）CsCS（MH_048698.1）、川桑（Morus notabilis C. K. Schneid.）MnCs（XP010087965.1）、红掌（Anthurium andraeanum Linden.）AaCS（JAT55223.1）亲缘关系最近，相似性分别达到86.49%、97.00%、86.00%。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的表达峰值时间，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，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，其中与AheCS2相关性显著。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，可作为进一步研究波罗蜜AheCS基因的功能及遗传改良的候选基因。
- 波罗蜜 /
- 柠檬酸 /
- AheCS基因 /
- 克隆 /
- 基因表达
Abstract: Objective 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 AheCS1, AheCS2, and AheCS3 from fruits of A. heterophyllus Haida 2 were cloned for a bioinformatic analysis. At room temperature(22±1℃) and 90% RH, changes on the gene expression and citric acid content in the fruits under natural ripening process or exposed to either 0.5 mg·L⁻¹ of 1-MCP or 1,000 mg·L⁻¹ of 40% ethylene (ETH) were determined. Result The citric acid content in a naturally ripening jackfruit gradually rose and declined subsequently. It increased at an accelerated rate when exposed to ETH, but the rate was slowed down by the 1-MCP treatment. The ORFs of the three genes ranged from 1 422 bp to 1 827 bp containing conserved WPNVDAHS domain and belonging to the CS family. The amino acid sequences were phylogenetically closely related to those of CsCS (MH_048698.1) in citrus with a similarity of 86.49%, MnCS (XP010087965.1) in mulberry with a similarity of 97%, and AaCS (JAT55223.1) in anthurium with a similarity of 86%. The expressions of these genes were low in the early stage and raised subsequently during natural ripening (CK). However, the exogenous ETH hastened the increasing rate of AheCS1 expression and elevated the levels of AheCS2 and AheCS3 expressions, while 1-MCP delayed the rise but heightened the expression levels of the three genes at the stage near fruit maturity. The citric acid content of the ripening fruits generally positively correlated with the gene expressions. The correlation with AheCS2 reached a statistically significant level. Conclusion AheCS2 is a potential gene involved in the regulation of citric acid accumulation during the ripening process of jackfruit, and it can be a candidate gene for further study of the function and genetic improvement of the AheCS gene in jackfruit .
- Jackfruit /
- citrate acid /
- AheCS /
- cloning /
- gene expression

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图 1 不同处理对柠檬酸含量的影响

不同字母表示差异显著（P<0.05）。

Figure 1. Effect of various treatments on citric acid in jackfruit

Data with different letters indicate significant difference at (P<0.05).

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图 2 AheCS1, AheCS2, AheCS3基因的克隆

M：DL2 000 DNA Marker；CS1/2/3: 波罗蜜目的基因。

Figure 2. Cloning of AheCS1, AheCS2, and AheCS3

M: DL2 000 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. Predicted tertiary structure of protein encoded by AheCS1, AheCS2, and AheCS3

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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 amino acid sequences of AheCS1, AheCS2, and AheCS3 proteins

Co: Camellia oleifera Abel.; Jc: Jatropha curcas L.; Mn: Morus notabilis C. K. 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.; Pas: Paeonia × suffruticosa Andr.; 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. Conserved motifs of AheCS1, AheCS2, and AheCS3 proteins

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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 of CSs in various species

St: Solanum tuberosum L.; Cf: Capsicum frutescens L. ; Cc: Coffea canephora Pierre ex Froehn.; Co: Camellia oleifera.; As: Armeniaca sibirica (L.) Lam.; Ps:Paeonia × suffruticosa Andr.; 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 expressions of AheCS1, AheCS2, and AheCS3

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表 1 AheCS1、AheCS2和AheCS3基因全长引物序列

Table 1. Sequences of AheCS1, AheCS2, and AheCS3 primers

引物名称 Primer names	引物序列（5′-3′） Primer sequences
AheCS1-F AheCS1-R AheCS2-F AheCS2-R AheCS3-F AheCS3-R	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. Sequences of quantitative qRT-PCR primers

引物名称 Primer names	引物序列（5′-3′） Primer sequences
AheCS1-F	5′-AGAATCAAGCACTAAGGGACG-3′
AheCS1-R	5′-TTCAGGAATTTGTGGAGGC-3′
AheCS2-F	5′-GCCTCCCATCCTAACAGAAA-3′
AheCS2-R	5′-CGCTCGGTCCCATACTAACT-3′
AheCS3-F	5′- CCAACCGAGTTCTTCCCTG-3′
AheCS3-R	5′- GATAATGCCGCAACCAAAC-3′
GAPDH-F	5′- TTGAAGGGTGGNGCNAARAARG -3′
GAPDH-R	5′- ATAACCCCAYTCRTTRTCRTAC-3′

下载: 导出CSV

表 3 AheCS1/2/3 蛋白的理化性质

Table 3. Physiochemical properties of AheCS1, AheCS2, and AheCS3 proteins

基因名称 Gene name	氨基酸数 Number of amino acids	分子质量 Molecular weight/kD	等电点 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 Top 3 amino acids/%
AheCS1	608	151.18	4.96	25.29	45.73	0.698	Ala 25.3 Gly25.7 Thr27.5
AheCS2	473	52.89	7.20	26.37	40.34	0.652	Gly24.6 Ala26.4 Thr29.7
AheCS3	513	56.42	8.15	25.02	47.61	0.748	Ala25.0 Gly25.4 Thr26.3

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表 4 AheCS1/2/3 蛋白的亚细胞定位预测

Table 4. Predicted subcellular localization of AheCS1, AheCS2, and AheCS3 proteins

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

下载: 导出CSV

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, AheCS2, and AheCS3 proteins

基因名称 Gene name	糖基化位点数 Number of glycosy/个	磷酸化位点数 number of phosphorylation sites/个			二级结构组分 secondary structure component/%
基因名称 Gene name	糖基化位点数 Number of glycosy/个	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

下载: 导出CSV

表 6 柠檬酸含量与AheCS1/2/3 基因表达的相关性分析

Table 6. Correlation between organic acids and AheCS1, AheCS2, and AheCS3

指标 Index	柠檬酸 Citrate acid	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。 * indicates significant correlation at 0.05 level (two-tailed).

下载: 导出CSV

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