Cloning and function analysis of GA signal pathway gene LcPIF4 in litchi

Abstract:【Objective】Phytochrome-interacting factors (PIFs) family proteins are a small subset of basic helix-loop-helix transcription factors that interact specifically with the active form of phytochrome (phy) photoreceptors and play an important role in GA signaling. PIFs control diverse aspects of plant growth and development, such as hypocotyl elongation, cotyledon growth, circadian rhythms, seed germination, high temperature tolerance and anthocyanin synthesis. LcPIF4, a homolog of Arabidopsis PIFs was identified in Litchi from RNA-seq data of panicle. Our study showed that LcPIF4 was highly expressed in panicle. In order to explore the function of LcPIF4 in the uniconazole induced panicle growth inhibition, its sequence characteristics, gene function and expression pattern were analyzed by bioinformatics analysis, agrobacterium-mediated transformation and RT-qPCR.【Methods】The sequence of LcPIF4 was obtained from RNA-seq data and the full length ORF was predicted through Open Reading Frame Finder software (https://www.ncbi.nlm.nih.gov/orffinder/) . For gene cloning, total RNA was extracted from root, stem, leaf, female flower, male flower, pulp, pericarp and panicle. About 5 μg of total RNA was used to synthesize first-strand cDNA using the M-MLV reverse transcriptase (Life technologies) . The cDNA were used as a template for amplification of the open reading frame (ORF) of LcPIF4 with pair of gene-specific primer. To analyze the expression pattern of LcPIF4, semi-quantita-tive reverse transcriptase PCR (qRT-PCR) was conducted. The primer used for qRT-PCR was designed by Primer Premier 5.0, and qRT-PCR were performed on ABI 7500 Real-Time PCR System. LcACTIN was used as internal control. Relative expressions of all replicas of each sample were calculated by the2-△△Ctmethod. pCAMBIA2300 and pCAMBIA2300-YFP were used to construct overexpression and YFP-fused vectors, LcPIF4 was cloned into the pCAMBIA2300 and pCAMBIA2300-YFP vector by recombination using the p EASY-Uni Seamless Cloning and Assembly Kit (Trans Gen Biotech) respectively. The sequencing-confirmed pCAMBIA2300-YFP-LcPIF4 vectors were transformed into agrobacterium tumefaciens GV3101, and then selected A. tumefaciens GV3101 clones were grown overnight for Arabidopsis transformation using flower dipping method. To analyze hypocotyl length, plates containing Arabidopsis seeds were germinated and incubated vertically under 14/10 h light/dark cycle. observation and measurement were conducted 7 days after sowing. To monitor the subcellular localization of LcPIF4, transgenic Arabidopsis roots were checked for YFP signal of fusion protein after 7 days of sowing. To analyze the interaction between LcPIF4 and Lc DELLA-1, Lc DELLA-1 was inserted into the p GADT7 prey vector (GAL4 activation-domain; AD) , LcPIF4 was fused into the p GBKT7 bait vector (GAL4 binding-domain; DBD) , Prey and bait vectors were then introduced into AH109 yeast strain.Transformed colonies were selected on synthetic complete medium lacking Leu and Trp. Transformants were maintained on the same medium and transferred to synthetic medium without Ade, His, Leu and Trp.【Results】The ORF of LcPIF4 was 1 617 bp length and encoded a protein containing 539 amino acids. The protein sequence of LcPIF4 shared similarity of 42.7% with the PIF4 from Arabidopsis. sequence analysis showed that LcPIF4 protein contained a typical conserved APB motif at the N-terminal regions and a conserved HLH domain at the C-terminal regions, suggesting that LcPIF4 could have a typical function of PIF4 protein. Expression pattern of LcPIF4 was analyzed by qRT-PCR, expression level of LcPIF4 was higher in leaf, panicle, male flower, female flower than that in stem, pericarp, pulp and root. In addition, Uniconazole was able to inhibit the expression LcPIF4, five days and ten days after Uniconazole treatment the expression levels of LcPIF4 were only 1/3 and 1/5 of the control, respectively. To determine the roles of LcPIF4 in plant growth and development, More than 10 independent T1 transgenic lines were generated with the 35 S:LcPIF4 construct. Three homozygous lines were selected in the T3 generation to examine the phenotypes. Compared with the WT plants, the hypocotyl of Arabidopsis overexpressing LcPIF4 were much longer. The hypocotyl length of transgenic Arabidopsis were 8.3 mm on average, while the hypocotyl length of WT plants was only 2.1 mm on average. To determine the subcellular location of LcPIF4 protein, its ORF was fused to YFP and then, the fusion gene under the control of the 35 S promoter was transformed into Arabidopsis using an agrobacterium-mediated transformation. YFP fluorescence was observed in the roots of 35 S:LcPIF4-YFP seedlings using spinning disk confocal microscopy and it was found that LcPIF4-YFP was localized at nucleus, suggesting that LcPIF4 could be targeted to the nucleus of cells. PIFs could play an important role in the GA signal pathway, GA promotion of hypocotyl elongation would require PIFs. previous studies showed that PIFs participated in GA signaling through direct interaction with DELLA proteins. To confirm the interaction between LcPIF4 and Lc DELLA-1 in litchi, LcPIF4 was fused with the AD domain of the pGADT7 vector and LcDELLA-1 was fused with the BD domain of the p GBKT7 vector. The bait and prey vectors were co-transformed into yeast through LiAc-mediated yeast transformation method, and the interaction between LcPIF4 and Lc DELLA-1 was reconstructed.【Conclusion】In this study, a homolog of the phytochrome-interacting factor LcPIF4 was identified. LcPIF4 could promote the elongation of Arabidopsis hypocotyl. LcPIF4 protein was localized at nuclear. LcPIF4 was highly expressed in panicle and responsed to uniconazole at expression level.Yeast two hybrid assay showed LcPIF4 protein could interact with Lc DELLA-1.