Genome-Wide Identification of GAD Gene Family in Tomato and Expression Profiling after Ralstonia solanacearum Infection

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

Acta Horticulturae Sinica ›› 2026, Vol. 53 ›› Issue (4): 1157-1174.doi: 10.16420/j.issn.0513-353x.2025-0152

• Genetic & Breeding · Germplasm Resources · Molecular Biology • Previous Articles Next Articles

Genome-Wide Identification of GAD Gene Family in Tomato and Expression Profiling after Ralstonia solanacearum Infection

DAI Yuru, SUN Pingping, WANG Qiong^*()

College of Plant Protection，Yangzhou University，Yangzhou，Jiangsu 225009，China

Received:2025-12-25 Revised:2026-02-05 Online:2026-04-25 Published:2026-04-20
Contact: WANG Qiong

Abstract

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

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.

Figures/Tables 12

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

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

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

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

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

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

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

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

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

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

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

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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Genome-Wide Identification of GAD Gene Family in Tomato and Expression Profiling after Ralstonia solanacearum Infection

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