Characterization of an algal phosphomannose isomerase gene and its application as a selectable marker for genetic manipulation of tomato

Abstract

Establishing a transgenic plant largely relies on a selectable marker gene that can confer antibiotic or herbicide resistance to plant cells. The existence of such selectable marker genes in genetically modified foods has long been criticized. Plant cells generally exhibit too low an activity of phosphomannose isomerase (PMI) to grow with mannose as a sole carbon source. In this study, we characterized PMI from the green microalga Chlorococcum sp. and assessed its feasibility as a selectable marker for plant biotechnology. Chlorococcum sp. PMI (ChlPMI) was shown to be closely related to higher plants but more distant to bacterial counterparts. Overexpression of ChlPMI in tomato induced callus and shoot formation in media containing mannose (6 g/L) and had an average transformation rate of 3.9%. Based on this transformation system, a polycistronic gene cluster containing crtB, HpBHY, CrBKT and SlLCYB (BBBB) was co-expressed in a different tomato cultivar. Six putative transformants were achieved with a transformation rate of 1.4%, which produced significant amounts of astaxanthin due to the expression of the BBBB genes. Taken together, these findings indicate that we have established an additional tool for plant biotechnology that may be suitable for genetically modifying foods safely.

Keywords: Algae; Astaxanthin; BHY, β-carotene hydroxylase; BKT, β-carotene ketolase; Chl, Chlorococcum sp; LCYB, Lycopene β-cyclase; MS, Murashige and Skoog; PCR, Polymerase chain reaction; PMI, phosphomannose isomerase; PSY, phytoene synthase; Phosphomannose isomerase; RACE, Rapid amplification of cDNA ends; Tomato; Transformation; UPLC, Ultra-performance liquid chromatography; WT, wild type.

Fig. 1

Sequence alignment (a) and structures (b) of classic type I PMIs from Chlorococcum sp. and other species. The conserved YXDXNHKPE motif is boxed in red. Green arrows label four residues (Gln-111, Glu-138, His-113 and His-285). Accession numbers of the PMI: Solanum lycopersicum -XP_004233489.1; Homo sapiens-CAA53657.1; Candida albicans-CAA57548.1; Escherichia coli- NP_416130.3.

Fig. 2

Phylogenetic tree of type I PMIs. MEGA7 software was used to construct the tree using the Neighbor-Joining method with 1000 bootstrap replicates. The bootstrap support values showing the confidence level are given at the clade nodes as percentages.

Fig. 3

Regeneration of transformed Micro-Tom tomato expressing ChlPMI on MS medium with 6 g/L mannose. (a) Untransformed cotyledon explants. (b) Shoots and callus tissues generated from transformed cells. (c) Regenerated seedlings. Bar = 1.5 cm.

Fig. 4

PCR detection (a) and chlorophenol red assay (b) of regenerated tomato plants. M−100 bp DNA Ladder; WT-Micro-Tom cultivar; 1–10-regenerated lines; +-pBI-PMI plasmid. Ctr-negative control without leaf tissue.

Fig. 5

Growth status of WT and PMI-BBBB plants. Plantlets of WT (left) and PMI-BBBB (right) (a) were planted in a greenhouse at flowering (b), and fruiting (c). WT-Beta cultivar. Bar = 1.5 cm.

Fig. 6

Pigment analysis in leaves of WT and PMI-BBBB. UPLC analysis of pigments (a) and contents of main pigments (b) in tomato leaves of WT and PMI-BBBB. WT-Beta cultivar. Identified pigments of peaks: 1-astaxanthin; 2-ketolutein; 3-lutein; 4-chlorophyll; 5-β-carotene. Data represent average values from measurements of three individual tomato leaves. Significant differences were determined by Student's t-tests. The P-Values are indicated as follows: ∗P < 0.05, ∗∗P < 0.01.