<div>Medicago truncatula seeds were cultured and developed in Thua Thien Hue province, Vietnam, and they were used as materials for cloning a DHDPS gene with the encoding of the isozyme dihydrodipicolinate synthase (DHDPS) as well as optimizing the culture conditions for having the highest DHDPS gene expression in Escherichia coli BL21 StarTM (DE3) cells. The results revealed that the coding region of the DHDPS gene from M. truncatula was 100% similar with M. truncatula 4-hydroxy-tetrahydrodipicolinate synthase 2 (DHDPS2) submitted in NCBI (accession &nbsp;number: XM_013589555.2), coding for a long polypeptide of 307 amino acid with the molecular mass of about 33495 Da (Protein ID: XP_013445009.1). The DHDPS gene was successfully expressed in the Escherichia coli BL21 StarTM (DE3) cells with a pET200 / D-TOPO vector, and this produced the DHDPS2 protein with molecular masses of approximately 33.87 kDa (&raquo;33.5 kDa of DHDPS2 and 3.7 kDa of fusion fragment of pET 200/D-TOPO vector). The effects of the six different culture mediums of LB, SOB, SOC, YJ, HSG and TB, the induction times of 2h, 4h, 6h, 8h, 10h and 12h, and the inducer concentrations of 0.2 mM; 0.5 mM; 0.7 mM; 1.0 mM; 1.2 mM and 1.5 mM IPTG (Isopropyl &acirc;-D-1-thiogalactopyranoside) were also investigated for the purpose of optimising the expression of DHDPS2 in E. coli cells, and it was found that strong expression of recombinant DHDPS2 protein in E. coli. BL21 (DE3) cells occurred on the TB, HSG and YJ culture mediums after 8 hours with 0.2 mM inducible IPTG (BioRad).</div>

<div>Cloning and optimizing the expression of the DHDPS gene in the Medicago truncatula</div>

Background:&nbsp; Abiotic stresses such as salinity and drought significantly reduce plant productivity worldwide. Dihydrodipicolinate synthase (DHDPS), a key enzyme in lysine biosynthesis, may play a crucial role in stress adaptation. 
 
Methods: In this study, we examined the tissue-specific expression patterns of the DHDPS gene and its corresponding enzyme activity in Medicago truncatula exposed to salinity (200 mM NaCl) and osmotic (180 mM mannitol) stress. Hydroponically grown three-week-old plants were subjected to short-term (2 h) and prolonged (24 h) treatments. Gene expression was quantified using a rigorously optimized quantitative real-time PCR (qRT-PCR) protocol and data were analyzed using CFX Manager&trade; software. 
 
Result:&nbsp; Our findings revealed a pronounced tissue-specific dichotomy in DHDPS expression and enzyme activity: transcript levels were significantly upregulated in roots (up to ~12-fold), while they were concurrently downregulated in leaves (by up to approximately half) across both stress conditions. Notably, root expression peaked under short-term mannitol exposure, suggesting a rapid and robust metabolic response likely linked to lysine-derived protective compounds. In contrast, the downregulation observed in leaves may reflect an energy-conserving strategy or a shift in metabolic priorities under stress. This distinct organ-specific regulation underscores the complexity of plant stress adaptation and offers promising avenues for targeted engineering of crop resilience.

Tissue-specific Regulation of Dihydrodipicolinate Synthase (DHDPS) Activity and Gene Expression in Medicago truncatula under Abiotic Stress