Legume Research

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Research Article

Legume Research, volume 43 issue 3 (june 2020) : 326-331

Cloning and optimizing the expression of the DHDPS gene in the Medicago truncatula

Hoang Thi Kim Hong, Pham Thi Hong Trang, Dang Thanh Long, Nguyen Thi Quynh Trang
1Hue University of Sciences, Hue University, Vietnam.

  • Submitted26-02-2019|

  • Accepted29-03-2019|

  • First Online 24-05-2019|

  • doi 10.18805/LR-482

Cite article:- Hong Kim Thi Hoang, Trang Hong Thi Pham, Long Thanh Dang, Trang Quynh Thi Nguyen (2019). Cloning and optimizing the expression of the DHDPS gene in the Medicago truncatula. Legume Research. 43(3): 326-331. doi: 10.18805/LR-482.

ABSTRACT

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  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 (»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 â-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).

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