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The study aimed to investigate the functions of SmGAD gene in Salix matsudana in response to waterlogging stress，provide theoretical basis and candidate target genes for elucidating willow waterlogging tolerance mechanism and breeding willows tolerant to waterlogging.
Using one-year-old willow branches as materials，bioinformatic tools were employed to analyze physicochemical properties，conserved domains，protein domains，hydrophilicity/hydrophobicity，and promoter cis-acting elements of the SmGAD protein，and a phylogenetic tree was constructed. Real-time fluorescence quantitative PCR （qRT-PCR） was used to detect the expression changes of SmGAD gene. The SmGAD gene was cloned，and the prokaryotic expression vector pET32a-SmGAD and silencing vector pYL156-SmGAD were constructed.Virus-induced gene silencing （VIGS） technology was used to silence SmGAD，and the gene function was verified through waterlogging treatment. Meanwhile，prokaryotic expression was performed to induce protein expression，and the changes in bacterial culture OD600 nm under H2O2 stress were measured.
The coding sequence （CDS） of SmGAD gene in Salix matsudana was 1497 bp in full length，and SmGAD protein consisted of 498 amino acid residues，with a theoretical molecular mass of 56512.01 Da，a theoretical isoelectric point of 5.80，an aliphatic index of 90.80，an instability index of 35.82，and a grand average of hydropathicity hydrophobicity coefficient of -0.221. Phylogenetic analysis revealed that the SmGAD protein shared high similarity with GAD homologous proteins in six species，including Camellia sinensis L.，Oryza sativa L.，Arabidopsis thaliana L.，and Gossypium hirsutum L. Promoter cis-acting element analysis showed that the promoter region of SmGAD gene contained methyl jasmonate -responsive elements，auxin-responsive elements，light-responsive elements，drought-responsive elements，prolamin-metabolism-responsive elements，and so on. Real-time fluorescence quantitative PCR analysis results revealed that the expression trends of SmGAD gene differed among various willow varieties after waterlogging treatment. VIGS experiment results showed that the silenced lines exhibited more severer leaf abscission and decay under waterlogging stress，with substantial peroxidative damage to cell membranes，along with poor root growth. Prokaryotic expression experiments demonstrated that the SmGAD protein played an important role in scavenging H2O2.
SmGAD gene，a positive regulatory factor in regulatory network of Salix matsudana in response to waterlogging and hypoxia stresses，can enhance plant resistance to waterlogging stress through mechanisms such as alleviating oxidative stress damage. Prokaryo-tic expression experiments confirm that the SmGAD protein can scavenge H2O2.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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