Fig 1.
The recombinant expression of EcBAP in E. coli BL21(DE3) strain.
(A) induced at 37°C for 4 h; (B) induced at 25°C overnight. M: Protein marker; UI: the total lysate of uninduced bacterial cells; T: the total lysate of induced bacterial cells; P: the pellet of ‘T’; S: the supernatant of ‘T’; Arrow-head indicates the recombinant EcBAP protein. The original gel images of this figure (A, B) are available in S1 File.
Fig 2.
The recombinant EcBAP has an apparent Phi-oxidizing activity in vitro.
(A) The recombinant EcBAP was analyzed by SDS-PAGE and coomassie blue staining for purity assessment; (B) The recombinant EcBAP at the non-denatured status was visualized by native-PAGE and coomassie blue staining; (C) The Phi-oxidizing activity of recombinant EcBAP was qualitatively evaluated by native-PAGE gel activity staining in a consecutive reactant system composed of Phi and methyl green; (D) Ten individual reactions (#1–#10) were conducted for Pi/AM/MG-based spectrometric assay to quantitatively determine the Phi-oxidizing activity of recombinant EcBAP, and (E) The calculated Phi-oxidizing activity (μg Pi · μg-1 EcBAP) of all ten reactions were shown together, with the mean (±SD) marked in red (also see S2 File). Arrow-head indicates the recombinant EcBAP protein. The original gel images for this figure (A–C) are available in S1 File.
Fig 3.
Leaf explant regeneration test of EcBAP(Kan) transgenic tobacco under low to moderate Phi stress.
Small leaf pieces (0.5 cm × 0.5 cm) of WT and EcBAP(Kan) transgenic tobacco were pairwise laid on (A) MS (-Pi) or (B) standard MS medium, containing Phi of low to moderate concentrations (1, 2, 3 mM). After 7 days, 1 month, and even 2 months, the differentiation/regeneration status of these leaf explants under Phi stress were photo-recorded and compared between WT and EcBAP(Kan) transgenic tobacco.
Fig 4.
The Agrobacterium-infiltrated tobacco transformation of plant vector pET(EcBAP) under Phi selection.
Experiments were performed on (A) standard MS medium and (B) MS (Pi-) medium without Pi supply.
Fig 5.
EcBAP(Phi) transgenic tobacco has an evident Phi-resistance, as evaluated by seed germination and seedling growth under Phi stress.
The sterilized seeds of WT tobacco and EcBAP(Phi) transgenic lines (1, 2, 6) were sowed on standard MS or MS (-Pi) medium individually containing different concentrations (0.5, 1, 2 mM) of Phi as well as 100 mg·L-1 Kan. Their holding petri-dishes were placed (A) horizontally or (B) vertically in a plant growth chamber at normal cultivation conditions for 2 weeks, and then photo-recorded for comparison.
Fig 6.
Weed control simulation test by judging the competitive growth of EcBAP(Phi) transgenic tobacco versus weed and WT tobacco.
A seed mixture of WT tobacco, EcBAP(Phi) transgenic tobacco line (1, 2, or 6) and Tall fescue weed (at a ratio of 1:1:4) was evenly sowed on a matrix composed of perlite, vermiculite and little gravel, which was individually irrigated with 0.1 x MS (–P, +80 mg·L-1 Pi, or +120 mg·L-1 Phi). After 15 days of standard cultivation in greenhouse, the growth status of mixed seedlings was photo-recorded and compared. Under the condition of 120 mg·L-1 Phi, the growth-dominant tobacco seedlings were circled in blue, while those inhibited individuals were marked by red arrow-heads.
Table 1.
Primers used in this study.
Table 2.
MS-based culture medium used in different stages of Agrobacterium-mediated tobacco transformation under selection of Kan or Phi.