Expression and analysis of FT genes in pineapple flowering induced by ethephon

Abstract: 【Objective】The FT(Flowering Locus T) gene is a kind of flowering integron gene, which plays an important role in plant flowering. The current study on the FT gene does not distinguish between flower induction and flower development, probably because the two processes are tightly coupled. Therefore, the FT gene has been less studied in the flower induction stage. In order to further study the functional localization of FT genes during ethephon-induced pineapple flowering, in this study two genes were successfully cloned, AcFT1 and AcFT2, from'Tainong 16'pineapple and their expression pattern was analyzed.【Methods】The pineapple hybrid variety'Tainong 16'was cultivated in the Pineapple Greenhouse Planting Base of Meiting Village, Chengmai County, Hainan Province.40% ethephon(1 600 mg · L-1) diluted with 300 times was used to induce pineapple flowering. Samples were taken at 1 h, 3 h, 6 h, 1 d, 6 d, 10 d, 15 d, 22 d and 31 d after treatment, including roots, stems, mature and young leaves, shoot tips and other tissues. After the samples were frozen in the liquid nitrogen,they were stored in a refrigerator at-80 ℃ until use. Stem tips or inflorescence treated with ethephon for 0, 10, 15, 31 and 40 days were fixed in FAA fixative solution, and then dehydration, dyeing, transparency, wax dipping, embedding and slicing were carried out. The total RNA was extracted using the BioTeKe polysaccharide polyphenol plant RNA extraction kit, and the cDNA was reverse transcribed by the TaKaRa reverse transcription kit. By searching the pineapple genome database, the sequence information of two FT genes was obtained, and based on this information, primers were designed to amplify the pineapple FT gene cDNA. The sequencing results were searched using the BLAST online software program provided by NCBI; the AcFT gene coding region was analyzed by ORF Finder in NCBI;the protein amount and isoelectric point were predicted by online software ProtParam; the protein secondary structure was analyzed by GOV IV. The NCBI database Blastp was used to search the homologous protein sequences of different species of the gene protein sequence, and DNAMAN6.06 was used for multi-sequence homology comparison analysis, and the phylogenetic tree was constructed by MEGA6.0. Using reverse-transcribed cDNA of total RNA as a template and EF1 as an internal reference gene, real-time quantitative PCR analysis was performed. The data were analyzed by 2-ΔΔCtmethod for relative quantitative analysis.【Results】Sequence analysis indicated that the AcFT1 and AcFT2 genes encode 178 and 177 amino acids, respectively, both of which contained the PBP domain and had typical structural features of the PEBP family. GOR IV predicted that the ratios of random coil and α-helix of AcFT1 protein were 56.74% and 13.48%, respectively, and the proportion of extended chain was about 29.78%. The random coil and α-helix ratios of AcFT2 protein were 64.97% and 10.17%, and the proportion of the extended chain was about 24.86%. The physicochemical properties of protein sequences showed that the theoretical isoelectric points(pI) of AcFT1 and AcFT2 proteins were 5.03 and 6.96,respectively, and the unstable parameters( Ⅱ) of proteins were 47.55 and 43.21, respectively. The fat solubility indexes were 20.81 and 84.12, respectively. The AcFT1 protein may be a hydrophilic protein and the AcFT2 protein seemed to be a lipophilic protein. Homology analysis showed that the amino acid sequence encoded by pineapple AcFT1 had higher identity with Musa acuminata FT3 of 67%; the amino acid sequence encoded by AcFT2 had higher identity with the amino acid sequence encoded by barley FT, wheat FT and ryegrass FT3, which were 72%, 72%, and 75%, respectively. According to the paraffin section, it was found that when ethephon was not applied, the apex of pineapple had no obvious protuberance. Flower primordia were observed 10 days after ethephon treatment. After 15 days of ethephon treatment, the protuberance of shoot tips and the formation of new flower primordia could be clearly seen. After 31 days of ethephon treatment, with the further differentiation of flower primordia,the apical growth cone gradually protruded. Flower formation was observed 40 days after ethephon treatment. We speculated that the flower bud differentiation stage of pineapple'Tainong 16'from vegetative to reproductive growth might occur before and after 30 days of ethephon application. Real-time fluorescence quantitative PCR analysis showed that both AcFT1 and AcFT2 had tissue-specific expression and relatively high expression was detected in stems and leaves. After ethephon treatment, the expression of AcFT1 and AcFT2 in stems and leaves showed a reverse trend, in which AcFT2 was significantly up-regulated by ethephon, showing an upward and downward trend, while AcFT1 showed an upward and downward trend. And it decreased first and then rised. The expression level of AcFT2 in shoot tips increased significantly 1 day after ethephon treatment, and the relative expression level was 178 times higher than that of control, while the expression level of AcFT1 decreased significantly. At 31 days after ethephon treatment, the expression level of AcFT2 in shoot tips reached the maximum, which was about 408 times higher than that of control, but the expression level of AcFT1 was very low.【Conclusion】Two AcFT genes were cloned, and the AcFT2 gene was highly expressed in 1 d after ethephon treatment and subsequent flower bud differentiation, indicating that AcFT2 played an important role in the induction of pineapple flowering in response to exogenous ethephon signals.