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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

李思彤 李真琴 林婉彤 王俊宁

李思彤,李真琴,林婉彤,等. 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 [J]. 福建农业学报,2024,39(2):1−11
引用本文: 李思彤,李真琴,林婉彤,等. 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 [J]. 福建农业学报,2024,39(2):1−11
LI S T, LI Z Q, LIN W T, et al. Expression and Correlation Analysis of Citrate Synthase Gene (CS) in Pomelo and Citric Acid Accumulation [J]. Fujian Journal of Agricultural Sciences,2024,39(2):1−11
Citation: LI S T, LI Z Q, LIN W T, et al. Expression and Correlation Analysis of Citrate Synthase Gene (CS) in Pomelo and Citric Acid Accumulation [J]. Fujian Journal of Agricultural Sciences,2024,39(2):1−11

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

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

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

  • 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(AheCS1AheCS2AheCS3),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。
  • 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

    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

    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

    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.

    图  5  AheCS1/2/3 蛋白保守基序分析

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

    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.

    图  7  成熟过程中AheCS1、AheCS2AheCS3基因的表达分析

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

    表  1  AheCS1AheCS2AheCS3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.
    下载: 导出CSV

    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
    AheCS1608151.184.96(酸性)25.29
    45.73(不稳定)0.698Ala 25.3%
    Gly25.7%
    Thr27.5%
    AheCS247352.897.20(碱性)26.3740.34(不稳定)0.652Gly24.6%
    Ala26.4%
    Thr29.7%
    AheCS351356.428.15(碱性)25.0247.61(不稳定)0.748Ala25.0%
    Gly25.4%
    Thr26.3%
    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 AheCS1/2/3 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
    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
    AheCS1159552440.95%16.78%9.54%32.73%
    AheCS201710953.70%8.88%7.82%29.60%
    AheCS302891147.85%9.96%5.08%37.11%
    下载: 导出CSV

    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 LocationAheCS1AheCS2AheCS3
    细胞核 Nuclear0.040.030.00
    质膜 Plasma membrane0.020.120.00
    胞外 Extra-cellular0.000.000.00
    细胞质Cytoplasmic8.810.000.00
    线粒体 Mitochondria0.076.940.00
    细胞质内层 Endoplasm retic0.080.000.33
    过氧化物酶体Peroxisomal0.005.919.41
    高尔基体Golgi0.000.010.14
    叶绿体 Chloroplast0.660.000.12
    液泡 Vacuolar0.310.000.00
    下载: 导出CSV

    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*.
    下载: 导出CSV
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  • 收稿日期:  2023-10-11
  • 修回日期:  2023-12-23
  • 网络出版日期:  2024-03-28

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