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Molecular cloning and functional analysis of Three FLOWERING LOCUS T (FT) homologous genes from Chinese Cymbidium.

Huang W, Fang Z, Zeng S, Zhang J, Wu K, Chen Z, Teixeira da Silva JA, Duan J - Int J Mol Sci (2012)

Bottom Line: Alignment of the AA sequences revealed that CsFT, CgFT and CeFT contain a conserved domain, which is characteristic of the PEBP-RKIP superfamily, and which share high identity with FT of other plants in GenBank: 94% with OnFT from Oncidium Gower Ramsey, 79% with Hd3a from Oryza sativa, and 74% with FT from Arabidopsis thaliana. qRT-PCR analysis showed a diurnal expression pattern of CsFT, CgFT and CeFT following both long day (LD, 16-h light/8-h dark) and short day (SD, 8-h light/16-h dark) treatment.While the transcripts of both CsFT and CeFT under LD were significantly higher than under SD, those of CgFT were higher under SD.Our data indicates that CgFT is a putative phosphatidylethanolamine-binding protein gene in Cymbidium that may regulate the vegetative to reproductive transition in flowers, similar to its Arabidopsis ortholog.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of South China Agricultural Plant Genetics and Breeding, South China Botanical Garden, The Chinese Academy of Sciences, Guangzhou 510650, China; E-Mails: weitingpink@hotmail.com (W.H.); zmfang88@163.com (Z.F.); cymbidium1979@yahoo.com.cn (J.Z.); wu_kunlin@163.com (K.W.); duanj@scib.ac.cn (J.D.) ; Graduate University of the Chinese Academy of Sciences, Beijing 100049, China.

ABSTRACT
The FLOWERING LOCUS T (FT) gene plays crucial roles in regulating the transition from the vegetative to reproductive phase. To understand the molecular mechanism of reproduction, three homologous FT genes were isolated and characterized from Cymbidium sinense "Qi Jian Bai Mo", Cymbidium goeringii and Cymbidium ensifolium "Jin Si Ma Wei". The three genes contained 618-bp nucleotides with a 531-bp open reading frame (ORF) of encoding 176 amino acids (AAs). Alignment of the AA sequences revealed that CsFT, CgFT and CeFT contain a conserved domain, which is characteristic of the PEBP-RKIP superfamily, and which share high identity with FT of other plants in GenBank: 94% with OnFT from Oncidium Gower Ramsey, 79% with Hd3a from Oryza sativa, and 74% with FT from Arabidopsis thaliana. qRT-PCR analysis showed a diurnal expression pattern of CsFT, CgFT and CeFT following both long day (LD, 16-h light/8-h dark) and short day (SD, 8-h light/16-h dark) treatment. While the transcripts of both CsFT and CeFT under LD were significantly higher than under SD, those of CgFT were higher under SD. Ectopic expression of CgFT in transgenic Arabidopsis plants resulted in early flowering compared to wild-type plants and significant up-regulation of APETALA1 (AP1) expression. Our data indicates that CgFT is a putative phosphatidylethanolamine-binding protein gene in Cymbidium that may regulate the vegetative to reproductive transition in flowers, similar to its Arabidopsis ortholog.

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Phenotypic analysis of transgenic Arabidopsis thaliana plants (35S::CgFT) that ectopically expressed CgFT. (A) Screening transgenic A. thaliana T0 lines on 1/2 MS containing 50 μg/mL Kan. (B–D) Phenotype of WT and 35S::CgFT plants (line 7-1, T1) at different stages of 12 h L/12 h D. The green lines of A. thaliana are putative transgenic plants that are (B) 15-days-old; (C) 20-days-old; (D) 25-days-old; (E) Phenotype of WT and 35S::CgFT plants (line 7-3-5 T2) under 14 days long day (LD, 16 h L/8 h D) treatment; (F) Phenotype of WT and 35S::CgFT plants (line 7-3-5 T2) under 14 days short day (SD, 8h L/16 h D) treatment. Bar = 5 mm for A, C, D, E, F.
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f5-ijms-13-11385: Phenotypic analysis of transgenic Arabidopsis thaliana plants (35S::CgFT) that ectopically expressed CgFT. (A) Screening transgenic A. thaliana T0 lines on 1/2 MS containing 50 μg/mL Kan. (B–D) Phenotype of WT and 35S::CgFT plants (line 7-1, T1) at different stages of 12 h L/12 h D. The green lines of A. thaliana are putative transgenic plants that are (B) 15-days-old; (C) 20-days-old; (D) 25-days-old; (E) Phenotype of WT and 35S::CgFT plants (line 7-3-5 T2) under 14 days long day (LD, 16 h L/8 h D) treatment; (F) Phenotype of WT and 35S::CgFT plants (line 7-3-5 T2) under 14 days short day (SD, 8h L/16 h D) treatment. Bar = 5 mm for A, C, D, E, F.

Mentions: Since the three Cymbidium FTs have the same AA sequences, we then randomly chose only one (CgFT) for functional verification. CgFT driven by the CaMV 35S promoter was transformed into wild-type (WT) A. thaliana plants for functional analysis in order to explore whether Cymbidium FT could regulate the transition to flowering in A. thaliana. 35S::CgFT transgenic A. thaliana T0 plants were screened on half-strength MS [16] containing 50 μg/mL Kan using a protocol from Cheng et al. [17] (Figure 5A). In total, 45 independent 35S::CgFT transgenic A. thaliana T1 plants were obtained. All transgenic plants showed identical phenotypes by flowering earlier than WT plants (Figure 5B–D). These 35S::CgFT transgenic plants (Figure 5) flowered at about 15 days after sowing by producing about four rosette leaves (Table 1; Figure 5B). The flowering time of WT A. thaliana plants was more than 30 days and produced nine or ten rosette leaves (Table 1). When transgenic plants were exposed to LD or SD, flowering was obviously promoted under LD (Figure 5E), but was slower under SD (Figure 5F) and ectopic expression of cgFT in A. thaliana was consistent with the AtFT gene expression pattern in A. thaliana [18,19].


Molecular cloning and functional analysis of Three FLOWERING LOCUS T (FT) homologous genes from Chinese Cymbidium.

Huang W, Fang Z, Zeng S, Zhang J, Wu K, Chen Z, Teixeira da Silva JA, Duan J - Int J Mol Sci (2012)

Phenotypic analysis of transgenic Arabidopsis thaliana plants (35S::CgFT) that ectopically expressed CgFT. (A) Screening transgenic A. thaliana T0 lines on 1/2 MS containing 50 μg/mL Kan. (B–D) Phenotype of WT and 35S::CgFT plants (line 7-1, T1) at different stages of 12 h L/12 h D. The green lines of A. thaliana are putative transgenic plants that are (B) 15-days-old; (C) 20-days-old; (D) 25-days-old; (E) Phenotype of WT and 35S::CgFT plants (line 7-3-5 T2) under 14 days long day (LD, 16 h L/8 h D) treatment; (F) Phenotype of WT and 35S::CgFT plants (line 7-3-5 T2) under 14 days short day (SD, 8h L/16 h D) treatment. Bar = 5 mm for A, C, D, E, F.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3472752&req=5

f5-ijms-13-11385: Phenotypic analysis of transgenic Arabidopsis thaliana plants (35S::CgFT) that ectopically expressed CgFT. (A) Screening transgenic A. thaliana T0 lines on 1/2 MS containing 50 μg/mL Kan. (B–D) Phenotype of WT and 35S::CgFT plants (line 7-1, T1) at different stages of 12 h L/12 h D. The green lines of A. thaliana are putative transgenic plants that are (B) 15-days-old; (C) 20-days-old; (D) 25-days-old; (E) Phenotype of WT and 35S::CgFT plants (line 7-3-5 T2) under 14 days long day (LD, 16 h L/8 h D) treatment; (F) Phenotype of WT and 35S::CgFT plants (line 7-3-5 T2) under 14 days short day (SD, 8h L/16 h D) treatment. Bar = 5 mm for A, C, D, E, F.
Mentions: Since the three Cymbidium FTs have the same AA sequences, we then randomly chose only one (CgFT) for functional verification. CgFT driven by the CaMV 35S promoter was transformed into wild-type (WT) A. thaliana plants for functional analysis in order to explore whether Cymbidium FT could regulate the transition to flowering in A. thaliana. 35S::CgFT transgenic A. thaliana T0 plants were screened on half-strength MS [16] containing 50 μg/mL Kan using a protocol from Cheng et al. [17] (Figure 5A). In total, 45 independent 35S::CgFT transgenic A. thaliana T1 plants were obtained. All transgenic plants showed identical phenotypes by flowering earlier than WT plants (Figure 5B–D). These 35S::CgFT transgenic plants (Figure 5) flowered at about 15 days after sowing by producing about four rosette leaves (Table 1; Figure 5B). The flowering time of WT A. thaliana plants was more than 30 days and produced nine or ten rosette leaves (Table 1). When transgenic plants were exposed to LD or SD, flowering was obviously promoted under LD (Figure 5E), but was slower under SD (Figure 5F) and ectopic expression of cgFT in A. thaliana was consistent with the AtFT gene expression pattern in A. thaliana [18,19].

Bottom Line: Alignment of the AA sequences revealed that CsFT, CgFT and CeFT contain a conserved domain, which is characteristic of the PEBP-RKIP superfamily, and which share high identity with FT of other plants in GenBank: 94% with OnFT from Oncidium Gower Ramsey, 79% with Hd3a from Oryza sativa, and 74% with FT from Arabidopsis thaliana. qRT-PCR analysis showed a diurnal expression pattern of CsFT, CgFT and CeFT following both long day (LD, 16-h light/8-h dark) and short day (SD, 8-h light/16-h dark) treatment.While the transcripts of both CsFT and CeFT under LD were significantly higher than under SD, those of CgFT were higher under SD.Our data indicates that CgFT is a putative phosphatidylethanolamine-binding protein gene in Cymbidium that may regulate the vegetative to reproductive transition in flowers, similar to its Arabidopsis ortholog.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of South China Agricultural Plant Genetics and Breeding, South China Botanical Garden, The Chinese Academy of Sciences, Guangzhou 510650, China; E-Mails: weitingpink@hotmail.com (W.H.); zmfang88@163.com (Z.F.); cymbidium1979@yahoo.com.cn (J.Z.); wu_kunlin@163.com (K.W.); duanj@scib.ac.cn (J.D.) ; Graduate University of the Chinese Academy of Sciences, Beijing 100049, China.

ABSTRACT
The FLOWERING LOCUS T (FT) gene plays crucial roles in regulating the transition from the vegetative to reproductive phase. To understand the molecular mechanism of reproduction, three homologous FT genes were isolated and characterized from Cymbidium sinense "Qi Jian Bai Mo", Cymbidium goeringii and Cymbidium ensifolium "Jin Si Ma Wei". The three genes contained 618-bp nucleotides with a 531-bp open reading frame (ORF) of encoding 176 amino acids (AAs). Alignment of the AA sequences revealed that CsFT, CgFT and CeFT contain a conserved domain, which is characteristic of the PEBP-RKIP superfamily, and which share high identity with FT of other plants in GenBank: 94% with OnFT from Oncidium Gower Ramsey, 79% with Hd3a from Oryza sativa, and 74% with FT from Arabidopsis thaliana. qRT-PCR analysis showed a diurnal expression pattern of CsFT, CgFT and CeFT following both long day (LD, 16-h light/8-h dark) and short day (SD, 8-h light/16-h dark) treatment. While the transcripts of both CsFT and CeFT under LD were significantly higher than under SD, those of CgFT were higher under SD. Ectopic expression of CgFT in transgenic Arabidopsis plants resulted in early flowering compared to wild-type plants and significant up-regulation of APETALA1 (AP1) expression. Our data indicates that CgFT is a putative phosphatidylethanolamine-binding protein gene in Cymbidium that may regulate the vegetative to reproductive transition in flowers, similar to its Arabidopsis ortholog.

Show MeSH
Related in: MedlinePlus