关键词: 枳, SPL9和SPL13, 亚细胞定位, 荧光定量RT-PCR

Abstract: 【Objective】 This study aimed to clone two full length cDNA of SPL9 and SPL13 SBPs (SQUAMOSA promoter binding proteins) transcription factors from Poncirus trifoliata (L.) Raf. and construct expression vectors of SPL9 and SPL13 for subcellular location analysis. Real time PCR was used to determine the tissue expression patterns of SPL9 and SPL13 for analysis of the role of SPL9 and SPL13 during growth and development in adult P. trifoliata. 【Method】 Bioinformatics analysis and RACE technology showed that the complete cDNAs cloned, designated as Pt-spl9 and Pt-spl13, was 1 519 bp and 1 824 bp in length, respectively. The sequences were deposited in GenBank database with accession no. of FJ502237 and FJ502238. Recombinant plasmid 35S-GW-FJ502237/FJ502238-GFP was introduced into onion epidermal cells by the particle bombardment method with a PDS1000/He. Transformed cells were incubated for 24 h at 22℃ in the dark and green fluorescence was monitored under a laser scanning confocal microscope. The SYBR Green I Real-time qRT-PCR was employed to analyze the expression of Pt-SPL9 and Pt-SPL13 in young leaf, stem, root, bud flower, flower, fruit and other organs. 【Result】 Bioinformatics analysis showed that the cDNA of Pt-SPL9 and Pt-SPL13 had the recognition sites of microRNA156. The deduced amino acid sequence of Pt-SPL9 and Pt-SPL13 were 388 and 379 residues, which were 48.9 %, 42.5 %, 41.7 % identical, respectively, with SPL9 of Antirrhinum majus, Arabidopsis thaliana and Zea mays; and 40.8%, 38.1%, 35.8% identical respectively with SPL13 of Arabidopsis thaliana, SPL16 of Oryza sativa, and TGA1 of Zea mays, respectively. Pt-SPL9 and Pt-SPL13 and other plant SBPs have the same amino acid sequences that are highly conserved as the designed SBP domain and two-way nuclear localization signal. Subcellular localization results showed that the Pt-SPL9 and Pt-SPL13 were localized in the nucleus. SYBR Green I real-time quantitative RT-PCR results showed that the Pt-SPL9 and Pt-SPL13 were expressed ubiquitously in various organs and tissues, but the expression levels were different. The expression of Pt-SPL9 was highest in the stem, and lower in flowers and leaves, and lowest in the root, flower buds and young fruit; Pt-SPL13 was expressed highest in young fruit, and the expression levels in the stems and flower buds were high too. Its expression level in leaves was not high and those in flowers and roots were very low. 【Conclusion】 Transcription factor Pt-SPL9 and Pt-SPL13 all have nuclear localization function, Pt-SPL9 and Pt-SPL13 might play important roles in trifoliate orange development.

Key words: Poncirus trifoliata, SPL9 and SPL13, subcellular localization, real-time RT-PCR