Molecular cloning and functional characterization of genes associated with flowering in citrus using an early-flowering trifoliate orange (Poncirus trifoliata L. Raf.) mutant
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To isolate differentially expressed genes during the juvenile-to-adult phase transition of an early-flowering trifoliate orange mutant (precocious trifoliate orange, Poncirus trifoliata), suppression subtractive hybridization was performed. In total, 463 cDNA clones chosen by differential screening of 1,920 clones were sequenced and 178 differentially expressed genes were identified, among which 41 sequences did not match any known nucleotide sequence. Analysis of expression profiles of the differentially expressed genes through hybridization on customized chips revealed their expression change was associated with the phase transition from juvenile to adult in the mutant. Open reading frames of nine selected genes were successfully determined by rapid amplification of cDNA ends. Expression analysis of these genes by real-time RT-PCR showed that transcript levels of several genes were associated with floral induction and inflorescence development. Among these genes, HM596718, a sequence sharing a high degree of similarity with Arabidopsis EARLY FLOWERING 5 (AtELF5) was discovered. Real-time PCR and in situ hybridization indicated its expression pattern was closely correlated with floral induction and flowering of the mutant. Ectopic expression of the gene in Arabidopsis caused early flowering; however, its functional characterization is different than the role of AtELF5 observed in Arabidopsis. A yeast two-hybrid assay indicated that PtELF5 significantly interacted with DUF1336 domain of a hypothetical protein, which has not yet been functionally characterized in woody plants. These findings suggest that PtELF5 may be a novel gene that plays an important role during the early flowering of precocious trifoliate orange.
KeywordsExpressed sequence tags (ESTs) Flowering time Precocious trifoliate orange Suppression subtractive hybridization (SSH)
This research was supported financially by the National Natural Science Foundation of China (Grant nos. 30971973, 31071777, 30921002) and the Science Foundation of the Doctoral Discipline of the Chinese Ministry of Education (Grant no. 20090146110009), the Fundamental Research Funds for the Central Universities, the 863 Project of China (Twelfth five) and the Project of 948 from Ministry of Agriculture of China (Grant no. 2011G21).
- Diatchenko L, Lau YFC, Campbell AP, Chenchik A, Moqadam F, Huang B, Lukyanov S, Lukyanov K, Gurskaya N, Sverdlov ED, Siebert PD (1996) Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci USA 93:6025–6030PubMedCrossRefGoogle Scholar
- Hackett WP (1985) Juvenility, maturation, and rejuvenation in woody plants. Hort Rev 7:109–155Google Scholar
- Krajewski AJ, Rabe E (1995) Citrus flowering: a critical evaluation. J Hort Sci 70:357–374Google Scholar
- Li ZM, Zhang JZ, Mei L, Deng XX, Hu CG, Yao JL (2010) PtSVP, an SVP homolog from trifoliate orange (Poncirus trifoliata L. Raf.), shows seasonal periodicity of meristem determination and affects flower development in transgenic Arabidopsis and tobacco plants. Plant Mol Biol 74:129–142PubMedCrossRefGoogle Scholar
- Liang S, Wang X, Wan T (1999) Precocious trifoliate orange (Poncirus trifoliata L. Raf.) biology characteristic and its stock experiment. ZheJiang Citrus 16:2–4 (in Chinese)Google Scholar
- Lim MH, Kim J, Kim YS, Chung KS, Seo YH, Lee I, Kim J, Hong CB, Kim HJ, Park CM (2004) A new Arabidopsis gene, FLK, encodes an RNA binding protein with K homology motifs and regulates flowering time via FLOWERING LOCUS C. Plant Cell 16:731–740 (online version contains web-only data)PubMedCrossRefGoogle Scholar
- Pang XM, Wen XP, Hu CG, Deng XX (2006) Genetic diversity of Poncirus accessions as revealed by amplified fragment length polymorphism (AFLP). J Hortic Sci Biotech 81:269–275Google Scholar
- Pillitteri LJ, Lovatt CJ, Walling LL (2004b) Isolation and characterization of LEAFY and APETALA1 homologues from Citrus sinensis L. Osbeck ‘Washington’. J Am Soc Hortic Sci 129:846–856Google Scholar
- Theien G, Saedler H (1999) The golden decade of molecular floral development (1990–1999): a cheerful obituary. Dev Genet 25:181–193Google Scholar
- Zhang JZ, Ai XY, Sun LM, Zhang DL, Guo WW, Deng XX, Hu CG (2011) Transcriptome profile analysis of flowering molecular processes of early flowering trifoliate orange mutant and the wild-type [Poncirus trifoliata (L.) Raf.] by massively parallel signature sequencing. BMC Genomics 12(1):63PubMedCrossRefGoogle Scholar