Genome-wide identification of PEBP gene family and functional analysis of TFL1 gene in peach (Prunus persica)

Abstract: 【Objective】The objective of this study was to analyze the distribution, structure and evolution of PEBP (phosphatidyl ethanolamine-binding proteins) protein family in peach (Prunus persica) genome by bioinformatics. Moreover, the function of PpTFL1 gene belonging to PEBP family was also analyzed. This study provided the theoretical basis of regulation of flowering time by using molecular technique in peach.【Methods】The peach PEBP proteins were identified from peach whole genome in phytozome V12.1 database using six reported PEBP proteins in Arabidopsis (Arabidopsis thaliana). Several sorts of informatic software, including GSDS, Protparam, Expasy, DNAMAN, MEGA 6.0 and PlantCARE, were used to analyze the chromosome location, gene structure, physicochemical properties of protein, multiple sequence alignment, the phylogenetic tree and the cis-acting element of promoter in PpPEBP gene family, respectively. The PpTFL1 was cloned by RT-PCR and the relative expression ofPpTFL1 in different tissues of‘Zhongyoutao 14’was detected by quantitative real-time PCR (qRT- PCR). The overexpression vector of PpTFL1 was constructed and introduced into Arabidopsis using flo- ral dip method by Agrobacterium-mediated transformation. Different transgenic lines and the relative expression of PpTFL1 and AtLFY1 were verified by PCR and qRT-PCR, respectively. The flowering time and phenotype of five transgenic lines were observed.【Results】Five PEBP protein family genes were identified from the whole peach genome and unevenly distributed on chromosomes 2, 5, 6 and 7. PpPEBP proteins were all basic amino acids and hydrophobic proteins, four of which were unstable pro- teins except PpBFT. Five PpPEBPs owned similar gene structure, all composed of four exons and three introns. The PEBP consensus motif and critical amino acid [Tyr85(Y)/His88(H) and Gln140(Q)/Asp144 (D)], which are responsible for the function of FT/TFL1, were present in five PpPEBPs. The results of evolutionary analysis with other seven plant species showed that PpPEBPs could be divided into three subclasses, including TFL1-like, MFT-like and FT-like. Two monocotyledonous plant species, rice (Oryza sativa) and maize (Zea mays) tend to be clustered together, while PpPEBPs was clustered together with another five dicotyledonous plant species, including apple (Malus × domestica), grape (Vitis vinif- era), cotton (Gossypium raimondii), strawberry (Fragaria ananassa) and Arabidopsis. Meanwhile, the PpPEBPs and MdPEBPs were firstly clustered together due to peach and apple belonging to Rosaceae. The cis-acting elements of promoter analysis showed that five PpPEBPs promoters contained elements related to light signal, abscisic acid and gibberellin. And the number of light signal was the most. The results also indicated that PpPEBPs were affected by environmental conditions and several hormones, such as light, temperature, drought stress, gibberellin, abscisic acid, salicylic acid, etc.. The coding se- quences (CDS) of cloned PpTFL1 were 519 bp, which encoded 172 amino acids. The results of qRT- PCR showed that the expression of PpTFL1 was the highest in apex of‘Zhongyoutao 14’and signifi- cantly higher than that in other tissues. While the expression of PpTFL1 was the lowest and had no sig- nificant difference among flowers, young leaves and fruits. The tissue expression results indicated thatPpTFL1 expression was tissue- specific. Five transgenic Arabidopsis lines with PpTFL1 verified by PCR were obtained. L1 and L2 delayed flowering for 19 d compared to wild type, while L3, L4 and L5 failed to bolt after continuous cultivation for 10 d. qRT-PCR analysis of different transgenic lines indi- cated that PpTFL1 was indeed expressed in five transgenic plants, while the expression was significant- ly different among different plants. The relative expression of PpTFL1 was the highest in L4 and signifi- cantly higher than that in other plants, which was the lowest in L1. Moreover, the relative expression ofAtLFY1 in WT was obviously higher than five transgenic lines. And the relative expression of AtLFY1in L5 was lower than other four transgenic lines, which had no obvious difference. Based on the results of phenotype and qRT-PCR analysis of different transgenic lines, it was suggested that TFL was a key gene involved in repressing flowering and maintaining the inflorescence meristem by preventing the ex- pression of LFY.【Conclusion】Five PpPEBPs classified into TFL1-like, MFT-like and FT-like subclass- es were identified from the peach genome. PpTFL1 expression had tissue specificity. Overexpression ofPpTFL1 resulted in delayed flowering in Arabidopsis. It was suggested that PpTFL1 was involved in the regulation of flowering, which provided the theoretical basis of regulation of flowering time by us- ing molecular technique in peach.