柠檬蔗糖代谢关键酶基因家族鉴定及表达分析

Identification and expression analysis of key enzyme gene family in lemon sucrose metabolism

  • 摘要: 【目的】对柠檬蔗糖代谢关键酶基因家族成员进行鉴定,并分析其在蔗糖积累有明显差异的2个柠檬品种果实发育过程中的表达情况,为揭示柠檬蔗糖代谢关键酶在果实发育中的作用机制及柠檬与其他柑橘类果实蔗糖代谢分子机制提供理论依据。【方法】运用生物信息学方法鉴定柠檬蔗糖代谢关键酶[蔗糖合成酶(SUS)、蔗糖磷酸合成酶(SPS)、蔗糖转化酶(INV)]基因家族成员,对其基本理化性质、保守基序、基因结构、系统进化、蛋白质保守功能域、顺式作用元件和进化模式等进行全面分析。基于转录组数据分析柠檬蔗糖代谢关键酶基因家族成员在不同组织中的表达模式,并利用实时荧光定量PCR检测其在果皮和果肉发育过程的表达情况。【结果】从柠檬基因组共鉴定获得6个ClSUS基因家族成员(ClSUS1~ClSUS6)、4个ClSPS基因家族成员(ClSPS1~ClSPS4)和15个ClINV基因家族成员(ClINV1~ClINV15),其中ClSPSs基因编码的氨基酸数量最多,ClSUSs、ClSPSsClINVs基因编码的保守基序数量和种类大致相同,ClSUSs基因内含子数目为12~14个,ClSPSs基因的内含子数目为12~13个,ClINVs基因的内含子数量为1~11个。ClSUS基因家族成员和ClINV基因家族成员各分为3个亚家族,ClSPS基因家族分为2个亚家族。ClSUS6、ClINV1和ClINV8蛋白均扩张出新的结构域,依据结构域不同,ClINV蛋白家族成员分为2种不同水解酶类型。ClSUSs、ClSPSsClINVs基因启动子区域均含有激素响应、光响应、胁迫响应和生长发育调控元件。共25个基因不均匀地分布在染色体上,出现基因串联和片段复制现象。基于转录组数据分析,发现仅有21个基因表达,其中7个基因在柠檬不同组织中特异性表达。21个基因的相对表达量与葡萄糖、果糖和蔗糖含量呈不同程度相关性,其中有9个基因与柠檬葡萄糖、果糖和蔗糖含量呈明显正相关或负相关。实时荧光定量PCR检测结果显示,ClSUSs、ClSPSsClINVs基因整体上在果皮中的表达量均比果肉中的表达量高,在果皮中高表达的基因有ClSUS4、ClSPS2、ClINV14ClINV1,推测这些基因在柠檬果实可溶性糖积累过程中起重要调控作用。【结论】鉴定出的25个柠檬蔗糖代谢关键酶基因家族成员在理化性质与基因结构方面均存在明显差异,具有明显的组织表达特异性,部分成员如ClSUS4、ClSPS2、ClINV1ClINV14等是调控柠檬果皮和果肉可溶性糖含量差异的关键基因。

     

    Abstract: 【Objective】 The purpose of the study was to identify the members of the key enzyme gene family of lemon sucrose metabolism and analyze their expression during fruit development in two lemon cultivars with obvious differences in sucrose accumulation, so as to provide reference for revealing the role of lemon sucrose metabolic key enzyme in fruit development and the molecular mechanism of sucrose metabolism in lemon and other citrus fruits.【Method】 Bioinformatics methodwas used to identify the gene family members of the key enzymes of lemon sucrose metabolismsucrose synthetase(SUS), sucrose phosphatesynthase(SPS), sucrose invertase(INV), and their basic physicochemical properties, conserved motifs, gene structures, phylogeny, protein-conserved functional domains, cisacting elements and evolutionary mode were comprehensively analyzed. The expression patterns of the gene family members of key enzymes of lemon sucrose metabolism in different tissues were analyzed based on transcriptomic data. Real-time fluorescence quantitative PCR(qRT-PCR) was used to detect its expression during peel and pulp development.【Result】 A total of 6 ClSUS gene family members(ClSUS1-ClSUS6), 4 ClSPS gene family members(ClSPS1-ClSPS4) and 15 ClINV gene family members(ClINV1-ClINV15) were identified from the lemon genome, all of which were hydrophilic proteins, among which ClSPSs gene encoded the largest number of amino acids. The numbers and types of conserved motifs encoded by CISUSs, ClSPSs and ClINVs genes were roughly the same, with the number of introns in ClSUSs gene ranging from 12 to 14, the number of introns in ClSPSs gene ranging from 12 to 13, and the number of introns in ClINVs gene ranging from 1 to11. ClSUS gene family members and ClINV gene family members were each divided into 3 subfamilies, and ClSPS gene family was divided into 2 subfamilies. ClSUS6, ClINV1 and ClINV8 proteins all expanded into new structural domains, and ClINV protein family members were divided into two different hydrolase types according to different structural domains. ClSUSs, ClSPSs and ClINVs gene promoter regions all contained hormonal response, light response, stress response and growth and development regulatory elements, and a total of 25 genes were unevenly distributed on chromosomes, resulting in gene tandem and fragment replication phenomenon. Based on transcriptome data analysis, it was found that only 21 genes were expressed, of which 7 genes were specifically expressed in different tissues of lemon. The relative expression of 21 genes was correlated with glucose, fructose and sucrose contents to varying degrees, among which 9 genes were greatly positively or negatively correlated with the contents of lemon glucose, fructose and sucrose. The qRT-PCR results showed that the expression of ClSUSs, ClSPSs and ClINVs genes in the peel as a whole was higher than that in the pulp, and the genes with high expression in the peel were ClSUS4, ClSPS2, ClINV14 and ClINV1, which were speculated to play an important role in the accumulation of soluble sugars in lemon fruit.【Conclusion】 The 25 identified members of the key enzyme gene family members of lemon sucrose metabolism have obvious differences in physicochemical properties and gene structures, and have great tissue expression specificity, and some members such as ClSUS4, ClSPS2, ClINV1 and ClINV14 are the key genes regulating the difference of soluble sugar content between lemon peel and pulp.

     

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