Cloning, Expression And Promoter Analysis Of Flowering Locus T (FT) Homologue In Malus × Domestica

The transition from vegetative to reproductive phase is an important event in flowering plant lifecycle and is also an essential stage of fruit production for fruit trees. As a perennial woody plant, apple has a long juvenile period, during which no reproductive development occurs. In malus, the juvenile period ranges from 4 to 10 years and has hampered traditional breeding and genetics studies. For example, breeding an apple cultivar by traditional crosses has to be cost 10～20 years. Therefore, it is important to study on the molecular mechanisms of flowering in apple trees to accelerate the genetic improvement and promote the research process. On the other hand, understanding the molecular mechanisms of flowering can regulate fruit trees genetically to achieve the precociousness and increasing fruit production. Following locus T (FT) gene as a "florigen", one of the flowering-time genes, can activate floral meristem feature genes and induce flowering. The research on the function and expression regulation of FT gene can shed light on its role in the transition from vegetative to reproductive phase and the molecular mechanism of FT gene regulation flowering time in fruit trees. It also provides the basis for shortening the juvenile and improving varieties of apple trees by genetic engineering.1, Cloning and expression analysis of FT gene in Malus x domesticaTo investigate the functions of FT homologues in Malus x domestica, one of FT homolog was isolated in'Fuji'apple by PCR cloning and sequence analysis. The results show that the full-length cDNA consisted of 598 bp, included a complete open reading frame (ORF) and encoded a protein of 174 amino acids. It was named MdFT3 (GenBank accession number:GQ465756). In comparison, the putative MdFT3 protein showed 99.4 %,93.7%,96.6%,87.4%,89.1% identities with that encoded by FT/FTL of Malus x domestica (MdFT2), Malus x domestica (MdFTL), Prunus persica (PpFT), Populus detoides (PdFTLl) and Vitis vinifera (VvFT), respectively. Phylogenetic analysis was conducted using the complete amino acid sequences and generated an unrooted tree containing three major clades. MdFT3, together whith PpFT, PmFT, PnFT, CiFT and other FT homologues were clustered into the FT subfamily of PEBP. MdFT3 has closer evolutionary distance with PpFT and PmFT than that with PnFT, CiFT and CTRSFTL1.The expression pattern of MdFTS was studied using semi-quantitative RT-PCR. The results showed that MdFT3 was expressed in the all tested organs and tissues. The expression of MdFT3 in leaves decreased with their developing, and the expression in buds is higher than in leaves. However, the expression in seeds is very low. With the development of flowers, the expression of MdFT3 declined. It was expressed higher in anther and ovary than petal, calyx and filaments.2, Cloning and sequence analysis of FT promoter in Malus×domesticaPromoter is an important regulatory element at transcriptional level. In this study, the DNA sequence of FT gene was isolated by PCR from'Fuji'apple. The sequence consisted of 1620 bp, which included three introns and four extrons and encoded a protein of 174 amino acids. The putative FT protein showed 99% identities with FT/FTL of Malus×domestica. A sequence of 1784 bp upstream sequence of the FT gene was isolated by using genome walking technology and the whole sequence is 3250 bp after splicing the FT gene with the upstream region, which named MdFT-P (GenBank accession number:GQ148775). The upstream region was analysed by the PLACE and PlantCARE database. It was showed that the sequence included 5UTR Pyrich stretch motifs and the promoter characterization structures, such as TATA-box and CAAT-box, to ensure the exact origination of transcription. The sequence also contains ABRE, GARE-motif, CGTCA-motif, light response element and potential cis-acting elements. Therefore, it is presumed that the expression of MdFT-P gene could be regulated by Abscisic acid (ABA), Gibberellin acid (GA), Methyl jasmonate (MeJA) and light.3, Construction of plant expression vector harboring GUS gene driven by MdFT-P promoter and transient expression of MdFT-P gene promoterTo characterize the cis-regulatory elements of MdFT-P promoter in apple, five pairs of primer were designed to amplify the continuously deleted 5'-end of MdFT-P promote and introduced the corresponding restriction site on each primer 5'ends. The sequences of the 5'-regulatory region (-1～-167;-1～-300;-1～-787;-1～-1102;-1～-1585) were amplified by the five pair primers. The PCR products were digested by restriction enzymes and substituted 35S CaMV promoter, which drive theβ-glucuronidase (GUS) reporter gene in plant expression vector pYH4215. Thus, the vectors harboring continuously deleted 5'-end of MdFT-P promoter driving GUS reporter gene were constructed.The vectors were then transferred to Agrobacterium EHA105. Tobacco leaf explants were co-cultivated with the different Agrobacterium strains, respectively. The transient expression of GUS gene driven by the 5'-end continuously deleted MdFT-P promoter was conducted by histochemical assay. It was showed that the 5 deleted promoters all have activity, and the promoter activation was increased with the promoter sequence prolonged. The research provides information on the transcription regulation of FT gene by studying the activity of the 5'-end continuously deleted MdFT-P promoter and determining the structure of transcription regulation domain.