番茄GAD基因家族鉴定与青枯菌胁迫下的表达分析

doi:10.16420/j.issn.0513-353x.2025-0152

摘要/Abstract

摘要：

谷氨酸脱羧酶（glutamate decarboxylase，GAD）是催化谷氨酸发生不可逆脱羧反应产生γ-氨基丁酸（gamma-aminobutyric acid，GABA）的关键蛋白酶，在植物生长和抗逆过程中发挥着重要作用。为系统探究番茄GAD家族成员的潜在抗青枯病功能，在番茄基因组数据库中鉴定出5个GAD基因，分别命名为SlGAD1 ~ SlGAD5。利用生物信息学方法对这5个基因家族成员进行理化性质、进化关系、基因结构、共线性和启动子等综合分析，并运用实时荧光定量PCR（quantitative real-time PCR，qRT-PCR）技术结合转录组数据，对其组织特异性表达模式，以及经细菌与病原相关分子模式（pathogen-associated molecular patterns，PAMP）处理后的诱导表达模式进行分析。SlGAD分布于不同的染色体上；根据进化关系，GAD家族蛋白可分为4个亚族，番茄GAD家族成员分布在亚族Ⅰ、Ⅱ、Ⅳ中；SlGAD均具有较高的氨基酸序列保守性，且其羧基末端含有钙调蛋白结合位点和酶活自抑制位点；SlGAD2 ~ SlGAD4启动子区域含有响应防御与逆境胁迫的相关元件，但转录组数据显示，仅SlGAD3的表达水平受细菌及PAMP处理的显著诱导。青枯菌GMI1000侵染番茄植株后，SlGAD1 ~ SlGAD4的转录水平发生了不同的变化。进一步在烟草中瞬时过表达SlGAD并接种青枯菌Y45后，生物量统计结果显示，SlGAD1与SlGAD4能显著抑制青枯菌生长，初步证明这两个蛋白正调控番茄对青枯病的抗性。此外，组织特异性表达和亚细胞定位分析证实，相较于SlGAD1 ~ SlGAD4，SlGAD5在各组织中的表达水平较高；且所有番茄SlGAD均定位于细胞质中。上述结果为番茄GAD基因家族的抗青枯病功能研究及抗病育种实践提供了理论基础和新的遗传资源。

关键词: 番茄, 谷氨酸脱羧酶, 青枯病, 基因家族, 表达分析, 抗病性, 亚细胞定位

Abstract:

Glutamate decarboxylase（GAD）is a key enzyme that catalyzes the irreversible decarboxylation of glutamate to produce gamma-aminobutyric acid（GABA），playing a significant role in plant growth and stress resistance. To systematically investigate the potential function of the tomato GAD members in bacterial wilt resistance，this study identified five genes named SlGAD1 to SlGAD5 from the tomato genome database. Comprehensive bioinformatics analyses were conducted to characterize their physicochemical properties，evolutionary relationships，gene structures，collinearity，and promoter cis-regulatory elements. Furthermore，tissue-specific expression patterns and induced expression patterns after treatment with bacterial infections or pathogen-associated molecular patterns（PAMPs）were examined by integrating transcriptomic data with quantitative real-time PCR（qRT-PCR）. SlGAD genes are distributed across various chromosomes. Phylogenetic analysis classifies the GAD family proteins into four subgroups，with tomato GAD members found exclusively in subgroupsⅠ，Ⅱ and Ⅳ. All SlGAD proteins show a high degree of amino acid sequence conservation，containing both calmodulin-binding sites and autoinhibitory domains at their C-termini. The promoter regions of SlGAD2 to SlGAD4 contain cis-regulatory elements associated with defense and stress response；however，transcriptomic data indicate that only the expression of SlGAD3 is significantly induced by bacteria and PAMPs. After infecting tomato plants with Ralstonia solanacearum strain GMI1000，the transcript levels of SlGAD1 to SlGAD4 exhibited significant but varying degrees of change. Transient overexpression of SlGAD in tobacco followed by infection with Ralstonia solanacearum strain Y45 revealed that SlGAD1 and SlGAD4 significantly inhibited bacterial growth. These results suggest that SlGAD1 and SlGAD4 positively regulate tomato resistance to bacterial wilt. In addition，both tissue-specific expression and subcellular localization analyses revealed that SlGAD5 exhibited a higher expression level across various tissues compared to SlGAD1 to SlGAD4，and all SlGADs were localized in the cytoplasm. These results provide a theoretical foundation and novel genetic resources for investigating the GAD gene family in tomato against bacterial wilt and for breeding resistant cultivars.

Key words: tomato, glutamate decarboxylase, bacterial wilt, gene family, expression analysis, disease resistance, subcellular localization

戴宇如, 孙萍萍, 王琼. 番茄GAD基因家族鉴定与青枯菌胁迫下的表达分析[J]. 园艺学报, 2026, 53(4): 1157-1174.

DAI Yuru, SUN Pingping, WANG Qiong. Genome-Wide Identification of GAD Gene Family in Tomato and Expression Profiling after Ralstonia solanacearum Infection[J]. Acta Horticulturae Sinica, 2026, 53(4): 1157-1174.

图/表 12

表1 番茄SlGAD及内参基因的qRT-PCR引物序列

Table 1 qRT-PCR primer sequences for tomato SlGAD and reference genes

基因名称Gene name	正向引物（5′-3′）Forward primer	反向引物（5′-3′）Reverse primer
SlGAD1	GCAATTGCTGAGTTTTGGAAGA	CCTTCAAAACAACCATTAATCCTTCCC
SlGAD2	AAAAGAACAAGGAAACCGGATG	TACTTTTCACAAGAGGCAAACG
SlGAD3	TGCTAGGGAAGGATTACTTGTT	GAGGTGTAATGTAACATAGCAATCG
SlGAD4	TGAGCTTCAGAATCGATGTGTA	CAATATTGGGCTTGTCATGAGG
SlGAD5	TTATCTTGGTGCTGATCAACCT	TAACCCTCATAACCCAAACGAA
SlUbiquitin7	GATCGTATCAAGGAACGTGTTG	CTTTAGCTGTCTTGTCATCTGC

表2 番茄SlGAD表达载体构建所用引物序列

Table 2 Primers for constructing tomato SlGAD expression vectors

基因名称Gene name	正向引物（5′-3′）Forward primer	反向引物（5′-3′）Reverse primer
SlGAD1	ATTTACGAACGATAGGGTACCATGGTTCTCTCAAAAACTCCT	GCCCTTGCTCACCATGGATCCACAAATAGATGCTTTCCTAGC
SlGAD2	ATTTACGAACGATAGGGTACCATGGTGTTAACAACGACGTCG	GCCCTTGCTCACCATGGATCCGCAGACTCCGCTGGTCTTCTT
SlGAD3	ATTTACGAACGATAGGGTACCATGGTTCTCTCAAAAACTTCC	GCCCTTGCTCACCATGGATCCGCAAACAGCTGCTTTCCTAGC
SlGAD4	ATTTACGAACGATAGGGTACCATGGTCATATCCAAGGCCGCC	GCCCTTGCTCACCATGGATCCACATATTACATTTTTGGTCTTCTT
SlGAD5	ATTTACGAACGATAGGGTACCATGGTTCTGTCCAAGATAGCA	GCCCTTGCTCACCATGGATCCACAAACTCCCATAGTCTTATTC

图1 番茄GAD基因家族成员在染色体上的定位

Fig. 1 Chromosomal distribution of the GAD gene family in tomato

表3 番茄GAD家族蛋白的基本信息与亚细胞定位预测

Table 3 Basic information and subcellular localization prediction of GAD family proteins in tomato

基因名称 Gene name	基因组登录号 Accession No.	染色体分布 Chromosomal location	氨基酸数量 Number of amino acids	蛋白质分子量/Da Protein molecular weight	理论等电点 Theoretical pI	不稳定指数 Instability index	平均疏水性 GRAVY	亚细胞定位 Subcellular localization
SlGAD1	Solyc01g005000	1	484	55 180.54	5.83	32.58	-0.211	细胞质Cytoplasm
SlGAD2	Solyc03g098240	3	502	56 768.23	5.88	34.13	-0.226	细胞质Cytoplasm
SlGAD3	Solyc04g025530	4	493	56 162.38	5.27	36.85	-0.203	细胞质Cytoplasm
SlGAD4	Solyc05g054050	5	497	56 171.47	5.63	35.06	-0.228	细胞质Cytoplasm
SlGAD5	Solyc11g011920	11	503	56 957.36	5.44	32.70	-0.240	细胞质Cytoplasm

图2 番茄、马铃薯、茄子、本氏烟草、辣椒、拟南芥、亚洲棉和水稻GAD基因家族的系统进化树

Fig. 2 Phylogenetic tree of GAD gene family in Solanum lycopersicum，Solanum tuberosum，Solanum melongena，Nicotiana benthamiana，Capsicum annuum，Arabidopsis thalian，Gossypium arboreum and Oryza sativa

图3 番茄SlGAD的结构特征（A）、氨基酸序列（B）及C端序列（C）比对分析

Fig. 3 Comparative analysis of the structural features（A），amino acid sequences（B），and C-terminal sequences（C）of SlGADs

图4 番茄GAD基因家族种内共线性（A）及其与拟南芥和亚洲棉的种间共线性（B）分析

Fig. 4 Analysis of intraspecific collinearity（A）of the GAD gene family in Solanum lycopersicum and its interspecific collinearity（B）with Arabidopsis thaliana and Gossypium arboreum

图5 番茄GAD基因家族功能分析 A：SlGAD中部分顺式作用元件分布；B：细菌及PAMP处理后SlGAD的表达分析

Fig. 5 Functional analysis of GAD gene family in tomato A：Distribution of cis-acting elements in part of SlGADs；B：Expression analysis of SlGADs after treatment with bacteria and PAMPs

图6 接种青枯菌GMI1000不同时间点番茄SlGAD基因的相对表达水平以ubiquitin为内参，第0天数值为1，进行单因素方差分析（ANOVA），数据表示平均值 ± 标准差。*P < 0.05，**P < 0.01，n = 3。下同

Fig. 6 Relative expression levels of SlGAD genes in tomato at different time points after inoculation with Ralstonia solanacearum strain GMI1000 Using ubiquitin as the reference gene and the values at day 0 as 1，data were analyzed by one-way ANOVA and are presented as mean ± SD；*P < 0.05，**P < 0.01，n = 3. The same below

图7 在本氏烟草中过表达SlGAD对青枯菌Y45生物量的影响以GFP为阴性对照

Fig. 7 Effect of SlGAD overexpression in Nicotiana benthamiana on the biomass of Ralstonia solanacearum strain Y45 Using GFP as the negative control

图8 番茄GAD家族基因的组织表达（A）及qRT-PCR（B）分析

Fig. 8 Tissue-specific expression（A）and qRT-PCR analysis（B）of tomato GAD family genes

图9 番茄SlGAD-GFP融合蛋白在本氏烟草中的亚细胞定位分析 A：蛋白免疫印迹（Western blot）检测；B：亚细胞定位；C：SlGAD-GFP融合蛋白在本氏烟草中的分布。T：总蛋白，S：可溶性蛋白，M：膜蛋白

Fig. 9 Subcellular localization of tomato SlGAD-GFP fusion proteins in Nicotiana benthamiana A：Protein detection by Western blot；B：Subcellular localization；C：Distribution of the SlGAD-GFP fusion protein in N. benthamiana. T：Total protein，S：Soluble protein，M：Membrane protein

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