Abstract: Background and Objective: Ginkgo biloba is a precious medicinal plant and has a long juvenile phase and spends 15-20 years in the vegetative phase before turning to reproductive phases, which makes breeding and cultivation of Ginkgo especially challenging. The FRI gene can regulate the FLC gene which inhibits flowering and further causes the late flowering of G. biloba. Therefore, the cloning and analysis of FRI gene can regulate the flowering time of G. biloba. Materials and Methods: The GbFRI gene and the protein sequence were analyzed using the online website of National Center for Biotechnology Information (NCBI), ProtParam and bioinformatic software of Clustal X2.0, Vector NTI 11.5 and MEGA6. The expression of GbFRI gene in different tissues of G. biloba was studied by quantitative RT-PCR (qRT-PCR). Data were analyzed with one-way ANOVA using SPSS11.0 for Windows. Results: The full length cDNA of GbFRI gene was 1702 bp (GenBank accession no. KY662058) and the open reading frame (ORF) covered 1602 bp, which encoded a 534 amino-acid protein. The predicted protein showed that a FRI superfamily and contain coiled-coil domains in two positions (between amino acids 55-100 and 405-450, respectively). The expression analysis results displayed that the highest GbFRI expression was in the male flowers. The GbFRI expression was higher in female flowers, stems than in the roots and fruits. The lowest relative expression of GbFRI was in the leaves. Conclusion: The GbFRI gene was isolated and characterized, laying a foundation for further study of vernalization pathway in G. biloba.
INTRODUCTION
Ginkgo biloba is an ancient relict plant in belongs to Ginkgoaceae and Ginkgo. It is a precious tree species for ornamental, wood and medicinal purposes. The extract of G. biloba leaves is very complex. The most effective ingredients are flavonoids, terpene lactone compounds and polyisopreno1. These compounds can improve blood circulation, treat cardiovascular diseases and protect liver function2. However, a long juvenile phase (15-20 years) of G. biloba brings serious obstacles to the breeding of G. biloba varieties3. Therefore, the regulation of flowering is important from an agronomic perspective. However, the molecular mechanisms that regulate flowering are still poorly understood in G. biloba.
Plants have evolved complex mechanisms to control the initiation of flowering in response to environmental cues or endogenous signals4,5. Flowering time in Arabidopsis depended on resetting and regulation of FLC expression during reproductive development6,7. In vernalization pathway, FRI activated the MADS-box transcription factor, Flowering Locus C. (FLC), a major repressor of this switch regulating flowering time4,7. The FRI promoted high levels of FLC expression and thus inhibited flowering, the action of an active FLC allele depended on an active FRI allele8. In Arabidopsis, the expression level of FLC gene determined whether Arabidopsis belonged to annual or biennial or perennial plants9,10. While the FRI gene has a regulatory effect on the gene of FLC, the inactive FRI led to a low expression of the FLC, which caused Arabidopsis to exhibit an early flowering phenomenon. Winter-annual accessions have functional FRI, promoted FLC expression and delayed flowering until FLC was silenced by a prolonged period of cold.
At present, there have been reported on isolated of the FRI gene from some Brassica Species11 and Brassica oleracea12. The study found that the FRI-Ler allele induced high expression levels of FLC gene, while the high expression of FLC delayed the flowering time of A. thaliana, making A. thaliana susceptible to vernalization and requiring low temperature induction to flower13. A molecular marker for the vernalization gene FRI was established and a central domain conserved for FRI and related proteins was discovered11,14. Proteasome-mediated degradation of FRI modulated flowering time in Arabidopsis during vernalization15. Research showed that the interactions of strong and weak alleles of the genes FRI and FLC. in many cases determined the variations in time to flower.
So far, the cloning and expression of the FRI gene in G. biloba have not been studied. In order to study the key genes regulating the flowering time of G. biloba, the FRI gene was cloned and the expression level of GbFRI gene in different tissues of G. biloba was studied by qRT-PCR. The purpose was to find out the relationship between FRI gene and long juvenile phase of G. biloba. The study of the GbFRI gene will help elucidate the molecular mechanisms of G. biloba flowering time.
MATERIALS AND METHODS
Materials: The materials were planted in the Ginkgo Science and Technology Garden, Yangtze University, pickted from 31 year G. biloba cultivar “Jiafoshou”. Different tissues of ginkgo at different developmental stages, including roots, stems, leaves, male flowers, female flowers, fruits were collected before the end of May 2017. All samples were quickly frozen in liquid nitrogen and kept at -80°C until to use. Gel recovery kit (Agarose Gel DNA purification Kit Ver.4.0) RNA extraction kit (MiniBEST Plant RNA Extraction kit) reverse transcription kit (PrimeScript™ 1st Strand cDNA Synthesis Kit) and PCR reagents for ampicillin (AMP), pMD19-T cloning vector and Escherichia coli competent cell DH5 were purchased from TaKaRa, Dalian Bao Biotechnology Company. Shanghai Sangon Biological Engineering Company performed sequence and synthesis of the primers.
Cloning of the GbFRI gene: Total RNA was extracted using MiniBEST Plant RNA Extraction kit from female flowers of G. biloba. The specific primers GbFRI-up and GbFRI-down (Table 1) for amplification were designed according to the transcriptome sequencing data of G. biloba. The PCR system was 25 μL. The amplification program was 94°C for 3 min; 32 cycles of 94°C for 30 sec, 56.5°C for 30 sec, 72°C for 1 min; a final extension at 72°C for 10 min. When the PCR product was successfully tested with 1% agarose gel electrophoresis, the target fragment was recovered according to the instructions of the gel recovery kit. After that, the target gene fragment was ligated into the pMD19-T vector and transformed into Escherichia coli strain DH5α. A single colony was picked and cultured. Screened positive clones were sent to Shanghai Sangon Biotech for sequencing.
| Table 1: | Primer sequences in this study |
Quantitative real-time PCR analysis: Total RNA was extracted from roots, stems, leaves, male flowers, female flowers, fruits of G. biloba. First-strand cDNA was synthesized by using PrimeScript™ RT reagent kit with gDNA Eraser (Perfect Real Time) (TaKaRa, Dalian, China). The primers GbFRI-FP and GbFRI-RP (Table 1) were designed for quantitative real-time PCR (qRT-PCR) amplification. GAPDH was used as the quantified internal reference gene. The upstream and downstream primers of GAPDH were GbGAPDH-FP and GbGAPDH-RP, respectively (Table 1). Referring to TaKaRa Company's AceQ® qPCR SYBR® green master mix (Without ROX) kit (Vazyme), real-time fluorescence was performed on Bio-Rad CFX. PCR programs were as follows: 95°C for 5 min, 40 cycles of 95°C for 10 sec, 60°C for 30 sec, 95°C for 15 sec, 60°C for 1 min and 95°C for 15 sec. Three repeated experiments at each sample. The relative expression fold of each sample was calculated by its Ct value normalized to the Ct-value of reference gene using the 2-ΔΔCt method described by Livak and Schmittgen16.
Statistical analysis: Data were analyzed with one-way ANOVA using SPSS 11.0 for Windows (SPSS Inc., Chicago, IL). The means were compared with Duncan’s multiple range tests. p-value of<0.05 was considered to be statistically significant.
RESULTS
Cloning and sequence analysis of GbFRI: The specific primers GbFRI-up and GbFRI-down designed by transcriptome sequencing data of G. biloba, amplified by PCR. The sequence analysis results showed that the full-length cDNA of FRI gene from G. biloba was 1,704 bp in length (Fig. 1). It contained a 1,604 bp ORF, encoding a putative protein of 534 amino acids (Fig. 1), namely GbFRI-1 (GenBank accession no. KY662058).
Analysis of GbFRI protein: Online analysis of ExPASy-ProtParam showed that predicted theoretical molecular weight and isoelectric point of GbFRI protein were 57.70 and 9.1 KDa, respectively. The analysis of phosphorylation sites using NetPhos 3.0 server indicated that many phosphorylation sites of serine and a few phosphorylation sites of threonine and tyrosine existed. The nucleotide sequence of the GbFRI gene exhibited highly homologous to the FRI gene sequences of other plants (Table 2) by BLAST-protein on NCBI, their similarities ranged from 42-60%.
The conserved functional domain of the GbFRI protein was analyzed online using BLAST (https://blast.ncbi. nlm.nih.gov/), which predicted that it had a conserved FRI superfamily domain. The GbFRI protein sequence was compared with FRI protein sequences of other plants (Fig. 2). The GbFRI protein had a highly conserved Frigida superfamily domain, indicating that GbFRI was a member of FRI gene family and was relatively conservative in evolution.
Molecular evolution analysis of GbFRI: To clarify the evolutionary relationship between GbFRI protein and FRI from other plants, the phylogenetic tree of FRI was constructed by CLUSTAL X2 and MEGA6 software using NJ method. As shown in the Fig. 3, the phylogenetic tree of FRI was divided into two major branches: Gymnosperms and Angiosperms. G. biloba was more closely related to gymnosperms than angiosperms. It was highly related to the Gymnosperms and G. biloba, which embodied the evolution of the GbFRI gene in the Gymnosperm. The FRI gene existed in Pinaceae, Gramineae, Arecaceae, Solanaceae and Leguminosae, which reflected the evolutionary diversity of the gene, which was consistent with the taxonomy of plant morphology.
Expression patterns analysis of GbFRI: The samples of G. biloba roots, stems, leaves, male flowers, female flowers, fruits were selected for qRT-PCR experiments to analyze further the specific expression of GbFRI in different tissues of G. biloba (Fig. 4).
| Table 2: | Protein sequence of GbFRI similarity to the FRI’s of other plant species |
| Fig. 1: | Nucleotide sequence and deduced amino acid sequence of GbFRI-1 |
| Start codon and termination codon are indicated by the box, the primers were indicated by the underline | |
The qRT-PCR results showed that GbFRI was expressed in roots, stems, leaves, male flowers, female flowers and fruits. The highest GbFRI expression was in the male flowers. Furthermore, the GbFRI expression was higher in female flowers and stems than in the roots and fruits. The lowest relative expression of GbFRI was in the leaves.
DISCUSSION
In Brassica, genomes A and C of FRI was represented by two loci: FRI.a and FRI.b17-19. Up to now, FRI in Brassica genome B was rarely investigated15. In Brassica genomes A and C, all FRIGIDA protein sequences contained the conserved central region corresponding to the Frigida domain characteristic for the superfamily of proteins FRIGIDA and FRIGIDALIKE19 1.
| Fig. 2: | Similarity analysis of GbFRI-coding protein and other known FRI proteins |
GbFRI : Ginkgo biloba (KY662058), EgFRI : Elaeis guineensis (XP_010930297.1), MaFRI: Musa acuminate (XP_009392592.1), JrFRI: Juglns regia (XP_018817942.1), NnFRI : Nelumbo nucifera (XP_010270166.1), MpFRI : Marchantia polymorpha (OAE19925.1), PpFRI : Physcomitrella patens (XP_001752883.1), PsFRI : Picea sitchensis (ABR17868.1), SmFRI : Selaginella moellendorffii (ABK252091) |
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| Fig. 3: | Phylogenetic tree of genes in plant FRI gene family |
| Expression patterns analysis of GbFRI | |
| Fig. 4: | Expression analysis of GbFRI in different tissues of G. biloba with qRT-PCR |
| Data are Means±SE for n = 3 biological replicates. Means with the different letters are significantly different at p<0.05 by Duncanʼs multiple range tests | |
Comparing the FRIGIDA sequences, the characteristic repetitive signatures were distinguished similar to the MEEKARSLS vernaliza repeat in FRI.a from A. thaliana. Genomes A had three MEEARSIS repeats and genomes C had two MEEARSIS repeats. Furthermore, MEGEARSIS and MQGEARSIS repeats in FRI.b from genomes A and C, respectively20. These repeats overlap form the Frigida domain and the coiled coil domain at the C-terminus of FRIGIDA protein14,17,18. In this study, the protein sequences of G. biloba had no repetitive signatures as them but found two α-helical coiled coil, which implied that GbFRI had a highly conserved FRIGIDA superfamily domain, this finding was consistent with the experimental result obtained in the present study. The result tentatively put forward that nucleotide and amino acid sequences of plants had mutated during the evolution, different plants might had different classification of the FRIGIDA gene. Each locus manifested genome specific polymorphisms, the FRIGIDA gene also possibly mutated in the evolution process and performing different functions. Whether the predicted coiled coils in the FRI protein were important for this function remains to be tested.
Consistent with previous studies that the expression of FRI gene existed in different tissues21. The qRT-PCR results indicated that GbFRI expression was higher in female flowers, stems than in the roots and fruits. The lowest relative expression of GbFRI was in the leaves. FRI promoted high expression levels of FLC, but the expression analysis of gene FLC in different organs and development periods have greater fluctuation22. The results may be due to the influence of FRI on FLC was regulated by season, temperature, different growth cycle or other factors.
CONCLUSION
In this paper, the FRI gene of G. biloba was coloned at the first time and found that a highly conserved FRIGIDA superfamily domain was contained in GbFRI protein, FRI gene was existed in different tissues in G. biloba. GbFRI plays an important role in flowering, this study will help researcher explore the long juvenile phase of G. biloba and develop Ginkgo industry in pharmacology, food, health care and other fields.
SIGNIFICANCE STATEMENTS
This study discovers the flowering regulation mechanism of GbFRI gene that can be beneficial for the development of ginkgo industry. The GbFRI is a key gene regulating the flowering time of G. biloba, which is useful for shortening the long juvenile phase of G. biloba through FRI gene silencing. The study will help the researcher to uncover the critical areas of FRIGIDA genes from G. biloba that many researchers were not able to explore. At the same time, it lays a theoretical foundation for shortening the infancy of woody plants by means of genetic engineering.
ACKNOWLEDGMENT
This work was supported by National Natural Science Foundation of China (No. 31670608).