C3HC4-type RING finger protein NbZFP1 is involved in growth and fruit development in Nicotiana benthamiana

Abstract

C3HC4-type RING finger proteins constitute a large family in the plant kingdom and play important roles in various physiological processes of plant life. In this study, a C3HC4-type zinc finger gene was isolated from Nicotiana benthamiana. Sequence analysis indicated that the gene encodes a 24-kDa protein with 191 amino acids containing one typical C3HC4-type zinc finger domain; this gene was named NbZFP1. Transient expression of pGDG-NbZFP1 demonstrated that NbZFP1 was localized to the chloroplast, especially in the chloroplasts of cells surrounding leaf stomata. Virus-induced gene silencing (VIGS) analysis indicated that silencing of NbZFP1 hampered fruit development, although the height of the plants was normal. An overexpression construct was then designed and transferred into Nicotiana benthamiana, and PCR and Southern blot showed that the NbZFP1 gene was successfully integrated into the Nicotiana benthamiana genome. The transgenic lines showed typical compactness, with a short internode length and sturdy stems. This is the first report describing the function of a C3HC4-type RING finger protein in tobacco.

Figure 1. Phylogenetic analysis of the relationships between NbZFP1 and C3HC4-type RING finger genes in other species.

We selected twenty-five typical C3HC4-type RING finger genes with high similarity from different species, and we analyzed the similarity of their RING finger domains and full-length sequences with NbZFP1. Phylogenetic trees were generated by the maximum likelihood (ML) method using MEGA4. Bootstrap values from 1000 replicates are indicated at each branch. (a) A maximum likelihood (ML) tree of NbZFP1 and genes from other species was constructed based on RING finger domain sequences. (b) A maximum likelihood (ML) tree of NbZFP1 and genes from other species was constructed based on full-length nucleotide sequences.

Figure 2. Purification and detection of recombinant protein.

The NbZFP1 protein was expressed in E. coli and purified with affinity chromatography (GSTrap™ HP column) and ion exchange chromatography (HiTrap Q HP column). The purified protein showed a single band on SDS-PAGE stained with Coomassie Brilliant Blue R-250. (a) Ion exchange chromatography of a concentrated solution loaded onto a HiTrap Q HP column. (b) SDS-PAGE analysis of the recombinant protein. Lane 1, GST protein after affinity chromatography purification. Lane 2, protein peak II as designated in (a). Lane 3, protein marker.

Figure 3. The results about subcellular localization of the NbZFP1 protein.

Laser-scanning confocal micrographs showing the fluorescence of leaf cells following infiltration with Agrobacteria carrying pGDG, pGDG-Rac1, or pGDG-NbZFP1 plasmids expressing GFP, Rac1-GFP, and NbZFP1-GFP proteins, respectively. Scale bar = 10 µm. (a) and (b) GFP expressed from pGDG; (c) and (d) GFP expressed from pGDG-Rac1; (e), (f), (g), and (h) Fluorescence expressed from pGDG-NbZFP1. (a), (c), (e), and (g) show the green channel; (b), (d), and (f) show an overlay of the bright-field and green channels; (h) shows an overlay of the bright-field, green, and red channels.

Figure 4. Characterization of VIGS strains by silencing of NbZFP1 compared with controls.

(a) Effect of VIGS on NbZFP1 (in this graph, NbA) transcription in N. benthamiana. TRV: The expression level of NbZFP1 in plants infected with TRV alone; TRV-A1: The expression level of NbZFP1 in plants infected with TRV-A1 (A1 is the C3HC4-type RING finger domain gene NbZFP1); TRV-A2: The expression level of NbZFP1 in plants infected with TRV-A2 (A2 is NbZFP1 lacking the C3HC4-type RING finger domain). (b) The phenotype of VIGS-silenced NbZFP1 and wild type strains. b1, NbZFP1-silenced plant at 20 days after the four-leaf stage; b2, wild type plant at 20 days after the four-leaf stage; b3, NbZFP1-silenced plant at 40 days after the four-leaf stage; b4, wild type plant at 40 days after the four-leaf stage. (c) The fruit phenotype of NbZFP1-silenced plants compared with controls. c1, c2, and c3: The fruit phenotype of non-silenced control plants. c4, c5, and c6: The fruit phenotype of NbZFP1-silenced plants.

Figure 5. Identification of transgenic tobacco plants.

Fresh leaves (100 mg) from T₁ plants were collected, and genomic DNA was extracted. A specific fragment of the gus gene (approximately 1000 bp in length) was amplified to identify the transgenic lines. (a) PCR identification of transgenic tobacco plants. M, DL8000 marker; 1, positive control; 2–10, transgenic tobacco lines. (b) Southern blot analysis of transgenic tobacco plants. 2, Transgenic line 2; 5, Transgenic line 5; 6, Transgenic line 6.

Figure 6. Characterization of T1 single copy lines compared with controls.

(a) The fruit phenotype of transgenic lines compared with wild type plants. a1 and a2, the fruit phenotype of transgenic plants. a3 and a4, the fruit phenotype of wild type plants. (b) The phenotype of T₁ single copy lines and wild type lines. b1, T₁ generation single copy transgenic strains at 20 days after the four-leaf stage; b2, wild type strains at 20 days after the four-leaf stage; b3: T₁ single copy transgenic strains at 50 days after the four-leaf stage; b4, wild type strains at 50 days after the four-leaf stage. (c) The height of single copy NbZFP1 transgenic plants compared with the height of wild type plants at different times. (d) The internode lengths of single copy NbZFP1 transgenic plants compared with the wild type internode length, 30 plants were measured. Asterisks indicate significant differences from the wild type: ^***P<0.001.