苹果柠檬酸合酶3基因家族成员鉴定及表达分析

Identification and expression analysis of citrate synthase 3 gene family members in apple

摘要:

柠檬酸合酶(citrate synthase 3, CS3)是细胞代谢途径中的关键酶之一，其活性调节着生物体的物质和能量代谢过程。本研究旨在从苹果全基因组中鉴定CS3基因家族成员，并进行生物信息学和表达模式分析，为研究苹果CS3基因的潜在功能提供理论基础。利用BLASTp基于GDR数据库鉴定苹果CS3家族成员，通过Pfam、SMART、MEGA5.0、clustalx.exe、ExPASy Proteomics Server、MEGAX、SOPMA、MEME和WoLF PSORT等软件分析CS3蛋白序列基本信息、亚细胞定位情况、结构域组成、系统进化关系以及染色体定位情况。利用酸含量的测定和实时荧光定量PCR (real-time fluorescence quantitative polymerase chain reaction, qRT-PCR)技术检测苹果6个CS3的组织表达和诱导表达特性。苹果CS3基因家族包含6个成员，这些CS3蛋白包括473−608个不等的氨基酸残基，等电点分布在7.21−8.82。亚细胞定位结果显示CS3蛋白分别定位在线粒体和叶绿体。系统进化分析可将其分为3类，各亚家族基因数量分别为2个。染色体定位结果显示，CS3基因分布在苹果不同的染色体上。蛋白二级结构以a-螺旋为主，其次是无规则卷曲，b-转角所占比例最小。筛选的6个家族成员在不同苹果组织中均有表达，整体表达趋势从高到低依次为MdCS3.4相对表达含量最高，MdCS3.6次之，其他家族成员相对表达量依次为MdCS3.3>MdCS3.2>MdCS3.1>MdCS3.5。qRT-PCR结果显示，MdCS3.1和MdCS3.3基因在酸含量较低的‘成纪1号’果肉中相对表达量最高，酸含量较高的‘艾斯达’果肉中MdCS3.2和MdCS3.3基因相对表达量最高。因此，本研究对不同苹果品种中CS3基因相对表达量进行了检测，并分析了其在苹果果实酸合成过程中的作用。结果表明，CS3基因在不同苹果品种中的相对表达量存在差异，为后续研究苹果品质形成机制提供了参考。

关键词:苹果;柠檬酸合酶(CS3)基因家族;生物信息学;柠檬酸;表达分析

Abstract:

As one of the key enzymes in cell metabolism, the activity of citrate synthase 3 (CS3) regulates the substance and energy metabolism of organisms. The protein members of CS3 family were identified from the whole genome of apple, and bioinformatics analysis was performed and expression patterns were analyzed to provide a theoretical basis for studying the potential function of CS3 gene in apple. BLASTp was used to identify members of the apple CS3 family based on the GDR database, and the basic information of CS3 protein sequence, subcellular localization, domain composition, phylogenetic relationship and chromosome localization were analyzed by Pfam, SMART, MEGA5.0, clustalx.exe, ExPASy Proteomics Server, MEGAX, SOPMA, MEME, WoLF PSORT and other software. The tissue expression and inducible expression characteristics of 6 CS3 genes in apple were determined by acid content and real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). Apple CS3 gene family contains 6 members, and these CS3 proteins contain 473-608 amino acid residues, with isoelectric point distribution between 7.21 and 8.82. Subcellular localization results showed that CS3 protein was located in mitochondria and chloroplasts, respectively. Phylogenetic analysis divided them into 3 categories, and the number of genes in each subfamily was 2. Chromosome localization analysis showed that CS3 gene was distributed on different chromosomes of apple. The secondary structure of protein is mainly a-helix, followed by random curling, and the proportion of b-angle is the smallest. The 6 members were all expressed in different apple tissues. The overall expression trend from high to low was the highest relative expression content of MdCS3.4, followed by MdCS3.6, and the relative expression level of other members was in the order of MdCS3.3>MdCS3.2>MdCS3.1>MdCS3.5. qRT-PCR results showed that MdCS3.1 and MdCS3.3 genes had the highest relative expression in the pulp of ‘Chengji No. 1’ with low acid content, and MdCS3.2 and MdCS3.3 genes in the pulp of ‘Asda’ with higher acid content had the highest relative expression. Therefore, in this study, the relative expression of CS3 gene in apple cultivars with different acid content in different apple varieties was detected, and its role in apple fruit acid synthesis was analyzed. The experimental results showed that the relative expression of CS3 gene in different apple varieties was different, which provided a reference for the subsequent study of the quality formation mechanism of apple.

Key words:apple;citrate synthase 3 (CS3) gene family;bioinformatics;citric acid;expression analysis