pDR195 vector

Manganese (Mn) and zinc (Zn) play important roles in plants. Members of the natural resistance-associated macrophage protein (NRAMP) family transport divalent metal ions. In this study, the function of peanut (Arachis hypogaea L.) AhNRAMP1 in transporting Mn and Zn and its potential for iron (Fe) and Zn bioaugmentation were examined. AhNRAMP1 transcription is strongly induced by manganese or zinc deficiency in peanut roots and stems. Yeast complementation analysis shows that AhNRAMP1 encodes a functional Mn and Zn transporter. Exogenous expression of AhNRAMP1 in tobacco and rice increases Mn or Zn concentrations and increases tolerance to Mn or Zn deficiency. AhNRAMP1 expression increased biomass of transgenic tobacco and rice, as well as yield of transgenic rice grown on calcareous soil. AhNRAMP1 contributes to manganese and zinc transport in plants and may be a candidate gene for iron and zinc biofortification.

Amplification of the full-length coding sequence of AhNRAMP1 with restriction sites. After digestion, AhNRAMP1 cDNA was introduced into the yeast expression vector pDR195. The resulting pDR195-AhNRAMP1 construct was then transformed into S. cerevisiae strains using the LiAc/SS-DNA/PEG method: the S. cerevisiae strains used were the Mn uptake-deficient mutant Δsmf1 and the Zn uptake-deficient mutant Δzhy6. The empty vector pDR195 was transformed into Δsmf1 and Δzhy6 as a negative control and into S. cerevisiae WT strain DEY1457 as a positive control. Yeast functional complementation experiments were performed using Δsmf1 (deficient in Mn uptake) and Δzhy6 (deficient in Zn uptake) yeast mutants to determine the Mn and Zn transport activities of AhNRAMP1. When grown on Mn- or Zn-limited medium, the growth of yeast mutants expressing AhNRAMP1 was significantly improved compared with yeast transformed with the empty vector (Figure 1). The results infer that AhNRAMP1 can complement Δsmf1 and Δzhy6 mutants and transport Mn and Zn.

Figure 1. Functional complementation of the Δsmf1 yeast mutant with AhNRAMP1 in Mn-limited medium (A), and functional complementation of the Δzhy6 yeast mutant with AhNRAMP1 in Zn-limited medium (B). Yeast mutants defective in Mn (Δsmf1) and Zn (Δzhy6) uptake transformed with the empty pDR195 vector were used as negative controls, and the WT yeast DEY1457 strain transformed with the empty pDR195 vector was used as a positive control. (Wang N, et al. 2019)