Cloning of GAD Gene in Pakchoi (Brassica campestris ssp. chinensis) and Induced Expression Analysis Treated with Exogenous GABA Under Higher Nitrogen Level
Abstract：Abstract Glutamate decarboxylase (GAD) mainly distributes in the cytoplasm and catalyzes the reaction of glutamate decarboxylation as a kind of intracellular enzyme found in many organisms. The metabolite of γ-aminobutyric acid (GABA) has the function of regulating plant growth, and development and improving the resistance to abiotic stress. This study cloned the gene of GAD in pakchoi (Brassica campestris ssp. chinensis) cultivar, cv. Wuyueman by the homologous cloning technology, namely BcGAD (GenBank accession No. KP852557). In addition, the effect of exogenous GABA on growth, expression of BcGAD gene and protein as well as enzyme activity were detected in pakchoi plants treated with application or soaking seeds of GABA under rhizosphere high nitrogen level. The results showed that the BcGAD gene contained a 1 485 bp ORF encoding 494 amino acids, which included a conserved domain of pyridoxal phosphate dependent aspartate aminotransferase superfamily. The homology analysis of amino acid sequence showed that the the similarity of amino acid sequences of BcGAD gene and GAD2 gene in B. rapa reached 100%, which demonstrated that there existed the closest homology between them. Moreover, the amino acid sequence of BcGAD was similar to GAD2 of B. napus, Raphanus sativus and B. juncea with the similarities of 99%, 99% and 98%, respectively. The treatments of soaking seeds at 3.75~7.5 mmol/L and application in nutrient solution at 1.25~5 mmol/L significantly promoted the growth supraterraneous compared with higher nitrogen treatment(P＜0.05). qRT-PCR and enzyme linked immunosorbent assay (ELISA) analysis showed that exogenous GABA increased the BcGAD gene transcriptional level and protein expression in pakchoi leaves treated with applying in nutrient solution and soaking seeds at different GABA concentration under the higher level of rhizosphere NO3-. BcGAD gene expression in leaves was significantly increased treated with soaking seeds (2.5~10 mmol/L) and applying GABA in nutrient solution (1.25~5 mmol/L) than that treated with high nitrogen condition. BcGAD protein expression in leaves enhanced accordingly treated with GABA of application in nutrient solution (3.75~10 mmol/L) and soaking seeds (1.25~5 mmol/L). Meanwhile, the GAD activities were significantly increasing by applying 5~6.25 mmol/L GABA in nutrient solution or soaking seeds with 2.5~3.75 mmol/L GABA correspondingly. In general, the treatments of soaking seeds at 3.75~6.25 mmol/L and applying solution at 2.5~3.75 mmol/L showed obvious effects among the different GABA concentration treatments(P＜0.05). Above all, the improved effects were dominant of soaking seeds at 5 mmol/L GABA and application GABA at 2.5 mmol/L. The results primarily confirmed that the growth, BcGAD, BcGAD protein and GAD activity were all affected by exogenous GABA concentration, application method and higher nitrogenlevel of pakchoi rhizosphere, which provides a theoretical basis for further utilization of BcGAD gene and exogenous GABA to improving the quality of leafy vegetables.
