辣木 MOGAD 基因克隆及表达分析.

[Objective] The MoGAD gene in Moringa oleifera was identified, and subsequent cloning, bioinformatics analysis, and expression level analysis were conducted. These investigations have established a solid foundation for exploring the functional relationship between the MOGAD genes and γ-aminobutyric acid (GABA) biosynthesis in M. oleifera. (Method) Using the GAD protein sequences of Arabidopsis thaliana (NP_197235.1) and Vitis vinifera (XP_002285268.1) to be as references, MOGAD family genes were identified from M. oleifera tran-scriptome data through local BLAST method and TBtools software. The genes were subsequently cloned using PCR technology. Bioinformatics online analysis websites and molecular docking technology were employed to analyze the physical and chemical properties, as well as the catalytic active sites of the MoGAD protein. Additionally, the real-time fluorescence quantitative RT-qPCR technology was utilized to assess the expression levels of MoGAD genes in different tissues. [Result] The four MoGAD genes were excavated from transcriptome data and cloned successfully. The length of the CDS region sequenced was 1494, 1212, 1470 and 942 bp respectively. MOGADI, MoGAD3 and MoGAD4 proteins had no transmembrane domains and belonged to extracellular proteins. The MoGAD2 gene had the transmembrane structure at amino acid positions 5-27 and 60-82, which encoded a kind of secreted proteins. The phylogenetic analysis showed that the largest number of motifs was in the branch of MoGADI gene family. The molecular docking revealed that four MoGAD proteins had binding sites for pyridoxal phosphate (PLP) and L-glutamate, and each protein had multiple amino acid residues that formed hydrogen bonds with PLP and L-glutamate. The results of fluorescence quantitative analysis showed that the expression levels of MoGADI and MoGAD2 genes in tender leaves, mature leaves and flowers were significantly higher than those of MoGAD3 and MoGAD4 genes, but the expression levels of these genes were relatively low in the stem, suggesting that MoGAD enzymes were mainly involved in GABA biosynthesis in the leaves of M. oleifera. Conclusion] MoGAD gene is successfully cloned from M. oleifera, and the physical and chemical properties of its encoded protein and the functional active sites of substrate binding are analyzed, which provide a theoretical basis for further study on the function of MoGAD genes. [ABSTRACT FROM AUTHOR]