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Abstract The cDNA of PHYC gene of tall fescue (Festuca arundinacea) was cloned by RT-PCR, named FaPHYC. The sequence was analyzed by bioinformatic software, and the results showed that the full-length cDNA(3816 bp) was obtained with an open reading frame predicated to be 3 411 bp, which encoded 1 136 amino acids. By BLASTP analysis, the N-terminal of FaPHYC was composed of GAF and pPhytochrome domains, and its C-terminal included two repeated PAS domains, one histidine kinase A domain and one histidine kinase-like ATPase domain. The phylogenetic analysis showed that FaPHYC was closely related to PHYCs from other Gramineae plants. The qRT-PCR results showed that FaPHYC expression was regulated by drought stress, heat stress, salt stress and nitrogen stress. The real-time quantitative PCR assay showed the expression of FaPHYC in tall fescue seedlings was continuously up-regulated during the light period and down-regulated in the dark. The results suggest that FaPHYC is photoperiod sensitive and plays a functional role in flowering regulation by photoperiod in tall fescue. The FaPHYC gene was cloned and reported from tall fescue for the first time, which lays a foundation for the investigation of the stress mechanism of FaPHYC in tall fescue plant.
Key words Festuca arundinacea; FaPHYC gene; Gene cloning; BIoinformatics analysis
Received: May 23, 2019 Accepted: August 28, 2019
Supported by project of High-level Innovative Talents Project in Guizhou Province (［2018］5634); Youth Fund of Guizhou Academy of Agricultural Sciences (2018-80); Guizhou Province S&T Contract Basics (［2019］1302).
Lu CAI (1987-), female, P. R. China, assistant researcher, devoted to research about pasture breeding and biotechnology.
* Corresponding author. E-mail: 1074253196@qq.com.
Plants are aware of changes in ambient light conditions through photoreceptors［1］. At present, there are four types of photoreceptors: phytochromes (PHYs), cryptochromes (CRYs), phototropins (PHOTs), and UV-B receptors［2］. The phytochromes in plants have two main functions［3］, one of which is to regulate the expression of photoresponsive genes through different signaling pathways, to affect the photomorphogenesis of plants［4-5］, and the other is to perceive the light information of surrounding environment, to participate in the biological clock to regulate flowering time［6-7］. Phytochromes regulate the whole growth and development process of plants from germination to maturity, and also participate in the circadian clock［8］. According to the stability of their proteins under red or white light, phyA is the only photolabile type, while phyB, phyC, phyD and phyE are all light stable［9-10］. Tall fescue (Festuca arundinacea) is one of the main turfgrass species used in China. As a kind of turfgrass, it takes a huge expense to prune it. Therefore, the phytochrome genes and their light signaling pathways in tall fescue are studied, so as to understand their regulation mechanisms in flowering of tall fescue and lay a foundation for cultivating the dwarf traits of tall fescue. Light is inextricably linked to the growth and development of plants. On the one hand, light provides indispensable energy to plants. On the other hand, plants can also respond to changes in light in the surrounding environment to adjust their own growth and development, to thereby adapt to the environment and optimize survival and reproduction［11］. Wheat phyC can activate the expression of PPD1, and then the flowering period of wheat is advanced under long-day conditions［12］. In addition, studies on barley and pearl millet have also shown that phyC is closely related to flowering stage［13-15］. PHYC plays an important role in plant photomorphogenesis and flowering regulation, but currently, no research on PHYC gene in tall fescue has been reported.
Tall fescue is a grassy plant of Festuca. It is a perennial cold-season pasture widely cultivated in temperate regions and can also be used as turfgrass. It has good heat and cold resistance and plays an important role in landscaping, turf establishment, environmental protection and soil and water conservation. It is also one of the main turfgrasses in China. In this study, with F. arundinacea Schreb. cv. ‘Qiancao No.1’ as a plant material, according to the known sequence spliced through sequencing of the transcriptome of tall fescue, the full-length cDNA of FaPHYC gene was cloned by rapid amplification of cDNA ends (RACE) and its expression was analyzed, laying a foundation for further research and application of this gene. Studying the resistance of tall fescue by molecular means will provide a theoretical basis for cultivating new tall fescue pastures or turfgrass varieties.
Materials and Methods
Plant material
F. arundinacea Schreb. cv. ‘Qiancao No.1’ (new national pasture variety, registration number: 299) bred by Institute of Prataculture, Guizhou Academy of Agricultural Sciences in 2005, was chosen as an experiment material. It was planted in the germplasm garden of Dushan Experimental Base, Guizhou Institute of Prataculture. The variety has the advantages of strong stress resistance, wide adaptability, good turf performance, good trampling resistance, good persistence, long green period, concentrated seed maturity and high yield, and is suitable for planting in low mountains and hills in the middle and lower reaches of the Yangtze River in China. It is an excellent grass species for lawn, environmental greening and ecological management.
Experiment methods RNA extraction and cDNA synthesis
According to the instruction of Trizol reagent (Invitrogen), the total RNA was extracted from the leaves of tall fescue. The total RNA after TE dilution was determined by OD at 260, 280 and 320 nm, respectively, and the RNA was detected by 1.0% agarose gel electrophoresis for integrity. A certain amount of the total RNA (2 μg) was subjected to reverse transcription using Oligo (dT) as a primer according to the instructions of RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas), to obtain the first strand cDNA.
Primer design and synthesis
According to the known sequence spliced through sequencing of the transcriptome of tall fescue in the early stage in laboratory, specific primers for PCR amplification were designed in the conserved domains of the gene, and they were used as specific primers for RT-PCR. Then, according to the specific sequence obtained by amplification, primers were designed using the software Premier 5.00. The primers for 5′ RACE were GSP1: AGCAGCACAATCACAT, GSP2: CATCCGCACTTTGTTCTT, GSP3: AACAGAAACCTGGACGCC, and the 5′ terminal was cloned. The primers were all sent to Shanghai Generay Biotech Co., Ltd. for synthesis.
Cloning of FaPHYC gene from tall fescue
PCR was performed with the cDNA of tall fescue as a template. The PCR system was 50 μl in volume, including cDNA 1 μl, 2×PCR Buffer for KOD FX Neo 25 μl, dNTPs (2 mmol/L) 10 μl, KOD FX Neo (1 U/μl) 1 μl, forward primer FaPHYC-fwd1 (10 μmol/L) 2 μl, reverse prime FaPHYC-rev1 (10 μmol/L) 2 μl, and ddH2O 9 μl. The PCR started with pre-denaturation at 98 ℃ for 5 min, followed by 30 cycles of denaturation at 98 ℃ for 10 s, annealing at 60 ℃ for 15 s and extension at 72 ℃ for 30 s, and completed with extension at 68 ℃ for 5 min. The PCR product was detected by 1.8% agarose gel electrophoresis, and the target band was rapidly cut under ultraviolet light. The target fragment was recovered according to the recovery kit (OMEGA) and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were analyzed, and the 3′ end of the FaPHYC gene was amplified according to the instructions of the 3′ RACE kit. The PCR product was recovered, and cloned and sequenced with T vector. The product was then ligated with known sequence using DNAMAN software, to obtain the full-length cDNA sequence of the gene.
Bioinformatics Analysis
The amino acid sequence homology of FaPHYC was analyzed by DNAMAN software. The online software BLAST of NCBI official website was used to perform search and alignment, and other amino acid sequences with high similarity were downloaded. A phylogenetic tree of FaPHYC was constructed and analyzed by DNAMAN software. The website ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) was used to find the open reading frame (ORF) of the gene. The physicochemical properties of the gene were predicted by the online software ProtParam in ExPASy Proteomics Server. The secondary structure, tertiary structure and protein signal peptides were predicted by SOPMA, SWISS-MODEL and SignalP, respectively. Real-time fluorescence quantitative PCR of FaPHYC from tall fescue
The tall fescue seeds were sown in a culture bowl with soil and cultured in a light incubator［light intensity 0.54 mmol/(m2·s), illumination time 12 h/d, temperature (24±2) ℃］for 30 d with routine management. Each treatment had 3 replicates. The normal young leaves were collected at intervals of 4 h in 48 h, and the collected leaves were frozen quickly with liquid nitrogen and stored in a refrigerator at -80 ℃. The total RNA was extracted according to the Trizol method, and cDNA synthesis was carried out according to the instructions of the reverse transcription kit. Real-time quantitative PCR was used to detect the expression of FaPHYC gene. According to the full-length cDNA sequence of FaFT2 gene, real-time fluorescent quantitative PCR primers were designed by BLAST program and synthesized by Shanghai Invitrogen. The real-time fluorescence quantitative PCR system was 10 μl, including 20 SYBR Premix Ex Taq (2×) 10 μl, upstream primer (10 μmol/L) 1 μl, downstream primer (10 μmol/L) 1 μl, cDNA (diluted 20 times) 2 μl, and ddH2O 8 μl. The reaction liquid was prepared on ice and placed on a TaKaRa PCR Thermal Cycler Dice Real Time System to allow reaction. The reaction was started with 95 ℃ for 2 min, followed by 45 cycles of 95 ℃ for 15 s and 55 ℃ for 15 s. Each sample was repeated 3 times. The relative expression level of the gene was calculated after the reaction was completed.
Results and Analysis
Extraction and detection of total RNA from tall fescue leaves
The total RNA extracted from tall fescue leaves was detected by electrophoresis on 1.0% agarose gel. The bands were clear and complete. The brightness of the 18S rRNA band was about half of that of the 28 S rRNA band (Fig. 1). The OD260/280 was determined by ultraviolet spectrophotometry to be 1.92, indicating that the extracted total RNA sample was of good quality and was not contaminated by degraded DNA, and could be used for subsequent experiment.
Cloning of full-length cDNA of FaPHYC gene from tall fescue
According to the known sequence spliced according to sequencing of the transcriptome of tall fescue in the early stage in laboratory, specific primers for PCR amplification were designed. With the cDNA of tall fescue as a template, PCR was performed, giving a gene fragment of about 600 bp. The PCR product was recovered, and ligated to T vector and sequenced, and an intermediate fragment of PHYC gene of tall fescue with a size of about 644 bp was obtained. The RACE-specific primers were designed using this sequence, and a 192 bp fragment of the 5′ end and a 213 bp fragment of 3′ end were obtained by 5′ RACE and 3′ RACE amplification, respectively. The full-length cDNA of PHYC gene of tall fescue was obtained after splicing. It was 3 816 bp in size (Fig. 3). The result of DNAMAN software analysis showed that the gene consisted of a 213 bp 3′ non-coding region, a 192 bp 5′ non-coding region, and an open reading frame of 3 411 bp in length, encoding 1 136 amino acid residues. It was designated FaPHYC. The BLAST online software (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to predict the conserved domains of the FaPHYC gene, which was found to have two conserved domains. The N terminus was composed of the GAF and phytochrome domains, and the C terminal included two repeated PAS domains, one histidine kinase A domain, and one histidine kinase-like ATPase domain.
Prediction of properties of the protein encoded by FaPHYC gene of tall fescue
The prediction was performed with the online software ProtParam. The results showed that the protein formula of FaPHYC gene was C5556H8870N1538O1673S59, which had the total number of atoms of 17 696 and the molecular weight of about 125 874.39 kDa. Its average hydrophilicity index, isoelectric point pI and instability coefficient were -0.147, 5.81 and 49.54, respectively. This protein was a hydrophilic protein (Fig. 5).
Characteristics of the protein encoded by FaPHYC gene of tall fescue
The structure of FaPHYC protein was analyzed by SOPMA online software. The results showed that α-helix, β-turn, random coil and extended strand of the secondary structure of the target protein accounted for 48.06%, 5.11%, 32.92% and 13.91%, respectively. The results of PSIPRED analysis showed that there were 35 α-helices and 35 β-sheets, and random coils connected α-helices with β-sheets (Fig. 6), which is consistent with the results of SOPMA analysis. The tertiary structure predicted by SWISS-MODEL software was identical to the secondary structure predicted by SOPMA and PSIPRED analysis (Fig. 7). The analysis results of the online software SignalP 4.1 indicated that the protein had no signal peptide sequence.
Analysis of FaPHYC gene expression in tall fescue
Real-time fluorescence quantitative PCR was used to analyze the expression of FaPHYC gene in leaves of tall fescue within 48 h day and night. The results showed that in the first photoperiod under light condition from 20:30 to 0:30, the level continued to increase, reaching the maximum value, while the expression level in the dark begun to decrease gradually, and decreased to the lowest at 04:30; and in the second photoperiod, the expression level continued to rise from 20:30 to 0:30 until the second peak, and then declined continuously. The highest peak of the FaPHYC gene expression can be detected in both photoperiods, and the peak of the first photoperiod was about 2.0 times of that of the second one. It indicated that the expression of FaPHYC gene was regulated by photoperiod, suggesting that this gene may play a certain role in the flowering pathway （Fig. 8）. Conclusions and Discussion
Studies in the model plant Arabidopsis thaliana indicate that the regulation of higher plant flowering is accomplished by the light signal transduction pathway and the genes involved in the circadian clock pathway such as GI (GIGANTEA), CO (CONSTANS) and FT (FLOWERING LOCUS T)［16-18］. The use of phytochrome genes to improve crops can reduce the degree of shade avoidance of crops and increase the density of crops to increase yield quickly and effectively, which has been confirmed in rice and potato research［19-20］. In addition, the maturity of crops can be changed to adapt to seasonal changes in different regions, and the sensitivity of crops to photoperiod can be reduced, thereby increasing the promotion area of short-day crops and long-day crops.
In recent years, the research on PHYC genes regulating photoperiod in plants has progressed rapidly, but there are still many scientific problems to be further explored. In this study, we analyzed the expression of FaPHYC gene and found that the highest peak of FaPHYC gene was detected in both photoperiods and the peak of the transcription level in the first photoperiod was greater than that in the second photoperiod, indicating that the gene is regulated by day and night illumination and affects plant flowering, which accords with the results of Kobayashi et al.［21］. Tall fescue ‘Qiancao No.1’ was bed by Wang et al.［22］from local wild tall fescue plants. It is planted in the short-day sunshine area of Dushan, Guizhou, and blooms for about 240 d, showing late flowering traits. In the long-day sunshine area, Altai, Xinjiang, it is an early-flowering phenotype.
In this study, the full-length cDNA of FaPHYC gene was obtained from tall fescue by 3′ RACE and 5′ RACE technology using tall fescue ‘Qiancao No.1’. It was found by amino acid alignment that this gene had higher similarity with Lolium perenne L., Aegilops tauschii Coss. and Brachypodium distachyon (L.) Beauv., which accords with the fact that they all belong to Gramineae. Protein domain and functional domain analysis indicated that the gene had two conserved domains, and its expression level was further analyzed. As for how the FaPHYC gene in tall fescue is regulated by light under different photoperiod conditions, it is necessary to further construct a vector to transform A. thaliana for functional verification.
References
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［15］NISHIDA H, ISHIHARA D, ISHII M, et al. Phytochrome C is a key factor controlling long-day flowering in barley［J］. Plant Physiology, 2013, 163(2): 804-814. ［16］SUREZ-LPEZ P, WHEATLEY K, ROBSON F, et al. CONSTANS mediates between the circadian clock and the control of flowering in Arabidopsis［J］. Nature, 2001, 410(6832): 1116-1120.
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Key words Festuca arundinacea; FaPHYC gene; Gene cloning; BIoinformatics analysis
Received: May 23, 2019 Accepted: August 28, 2019
Supported by project of High-level Innovative Talents Project in Guizhou Province (［2018］5634); Youth Fund of Guizhou Academy of Agricultural Sciences (2018-80); Guizhou Province S&T Contract Basics (［2019］1302).
Lu CAI (1987-), female, P. R. China, assistant researcher, devoted to research about pasture breeding and biotechnology.
* Corresponding author. E-mail: 1074253196@qq.com.
Plants are aware of changes in ambient light conditions through photoreceptors［1］. At present, there are four types of photoreceptors: phytochromes (PHYs), cryptochromes (CRYs), phototropins (PHOTs), and UV-B receptors［2］. The phytochromes in plants have two main functions［3］, one of which is to regulate the expression of photoresponsive genes through different signaling pathways, to affect the photomorphogenesis of plants［4-5］, and the other is to perceive the light information of surrounding environment, to participate in the biological clock to regulate flowering time［6-7］. Phytochromes regulate the whole growth and development process of plants from germination to maturity, and also participate in the circadian clock［8］. According to the stability of their proteins under red or white light, phyA is the only photolabile type, while phyB, phyC, phyD and phyE are all light stable［9-10］. Tall fescue (Festuca arundinacea) is one of the main turfgrass species used in China. As a kind of turfgrass, it takes a huge expense to prune it. Therefore, the phytochrome genes and their light signaling pathways in tall fescue are studied, so as to understand their regulation mechanisms in flowering of tall fescue and lay a foundation for cultivating the dwarf traits of tall fescue. Light is inextricably linked to the growth and development of plants. On the one hand, light provides indispensable energy to plants. On the other hand, plants can also respond to changes in light in the surrounding environment to adjust their own growth and development, to thereby adapt to the environment and optimize survival and reproduction［11］. Wheat phyC can activate the expression of PPD1, and then the flowering period of wheat is advanced under long-day conditions［12］. In addition, studies on barley and pearl millet have also shown that phyC is closely related to flowering stage［13-15］. PHYC plays an important role in plant photomorphogenesis and flowering regulation, but currently, no research on PHYC gene in tall fescue has been reported.
Tall fescue is a grassy plant of Festuca. It is a perennial cold-season pasture widely cultivated in temperate regions and can also be used as turfgrass. It has good heat and cold resistance and plays an important role in landscaping, turf establishment, environmental protection and soil and water conservation. It is also one of the main turfgrasses in China. In this study, with F. arundinacea Schreb. cv. ‘Qiancao No.1’ as a plant material, according to the known sequence spliced through sequencing of the transcriptome of tall fescue, the full-length cDNA of FaPHYC gene was cloned by rapid amplification of cDNA ends (RACE) and its expression was analyzed, laying a foundation for further research and application of this gene. Studying the resistance of tall fescue by molecular means will provide a theoretical basis for cultivating new tall fescue pastures or turfgrass varieties.
Materials and Methods
Plant material
F. arundinacea Schreb. cv. ‘Qiancao No.1’ (new national pasture variety, registration number: 299) bred by Institute of Prataculture, Guizhou Academy of Agricultural Sciences in 2005, was chosen as an experiment material. It was planted in the germplasm garden of Dushan Experimental Base, Guizhou Institute of Prataculture. The variety has the advantages of strong stress resistance, wide adaptability, good turf performance, good trampling resistance, good persistence, long green period, concentrated seed maturity and high yield, and is suitable for planting in low mountains and hills in the middle and lower reaches of the Yangtze River in China. It is an excellent grass species for lawn, environmental greening and ecological management.
Experiment methods RNA extraction and cDNA synthesis
According to the instruction of Trizol reagent (Invitrogen), the total RNA was extracted from the leaves of tall fescue. The total RNA after TE dilution was determined by OD at 260, 280 and 320 nm, respectively, and the RNA was detected by 1.0% agarose gel electrophoresis for integrity. A certain amount of the total RNA (2 μg) was subjected to reverse transcription using Oligo (dT) as a primer according to the instructions of RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas), to obtain the first strand cDNA.
Primer design and synthesis
According to the known sequence spliced through sequencing of the transcriptome of tall fescue in the early stage in laboratory, specific primers for PCR amplification were designed in the conserved domains of the gene, and they were used as specific primers for RT-PCR. Then, according to the specific sequence obtained by amplification, primers were designed using the software Premier 5.00. The primers for 5′ RACE were GSP1: AGCAGCACAATCACAT, GSP2: CATCCGCACTTTGTTCTT, GSP3: AACAGAAACCTGGACGCC, and the 5′ terminal was cloned. The primers were all sent to Shanghai Generay Biotech Co., Ltd. for synthesis.
Cloning of FaPHYC gene from tall fescue
PCR was performed with the cDNA of tall fescue as a template. The PCR system was 50 μl in volume, including cDNA 1 μl, 2×PCR Buffer for KOD FX Neo 25 μl, dNTPs (2 mmol/L) 10 μl, KOD FX Neo (1 U/μl) 1 μl, forward primer FaPHYC-fwd1 (10 μmol/L) 2 μl, reverse prime FaPHYC-rev1 (10 μmol/L) 2 μl, and ddH2O 9 μl. The PCR started with pre-denaturation at 98 ℃ for 5 min, followed by 30 cycles of denaturation at 98 ℃ for 10 s, annealing at 60 ℃ for 15 s and extension at 72 ℃ for 30 s, and completed with extension at 68 ℃ for 5 min. The PCR product was detected by 1.8% agarose gel electrophoresis, and the target band was rapidly cut under ultraviolet light. The target fragment was recovered according to the recovery kit (OMEGA) and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were analyzed, and the 3′ end of the FaPHYC gene was amplified according to the instructions of the 3′ RACE kit. The PCR product was recovered, and cloned and sequenced with T vector. The product was then ligated with known sequence using DNAMAN software, to obtain the full-length cDNA sequence of the gene.
Bioinformatics Analysis
The amino acid sequence homology of FaPHYC was analyzed by DNAMAN software. The online software BLAST of NCBI official website was used to perform search and alignment, and other amino acid sequences with high similarity were downloaded. A phylogenetic tree of FaPHYC was constructed and analyzed by DNAMAN software. The website ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) was used to find the open reading frame (ORF) of the gene. The physicochemical properties of the gene were predicted by the online software ProtParam in ExPASy Proteomics Server. The secondary structure, tertiary structure and protein signal peptides were predicted by SOPMA, SWISS-MODEL and SignalP, respectively. Real-time fluorescence quantitative PCR of FaPHYC from tall fescue
The tall fescue seeds were sown in a culture bowl with soil and cultured in a light incubator［light intensity 0.54 mmol/(m2·s), illumination time 12 h/d, temperature (24±2) ℃］for 30 d with routine management. Each treatment had 3 replicates. The normal young leaves were collected at intervals of 4 h in 48 h, and the collected leaves were frozen quickly with liquid nitrogen and stored in a refrigerator at -80 ℃. The total RNA was extracted according to the Trizol method, and cDNA synthesis was carried out according to the instructions of the reverse transcription kit. Real-time quantitative PCR was used to detect the expression of FaPHYC gene. According to the full-length cDNA sequence of FaFT2 gene, real-time fluorescent quantitative PCR primers were designed by BLAST program and synthesized by Shanghai Invitrogen. The real-time fluorescence quantitative PCR system was 10 μl, including 20 SYBR Premix Ex Taq (2×) 10 μl, upstream primer (10 μmol/L) 1 μl, downstream primer (10 μmol/L) 1 μl, cDNA (diluted 20 times) 2 μl, and ddH2O 8 μl. The reaction liquid was prepared on ice and placed on a TaKaRa PCR Thermal Cycler Dice Real Time System to allow reaction. The reaction was started with 95 ℃ for 2 min, followed by 45 cycles of 95 ℃ for 15 s and 55 ℃ for 15 s. Each sample was repeated 3 times. The relative expression level of the gene was calculated after the reaction was completed.
Results and Analysis
Extraction and detection of total RNA from tall fescue leaves
The total RNA extracted from tall fescue leaves was detected by electrophoresis on 1.0% agarose gel. The bands were clear and complete. The brightness of the 18S rRNA band was about half of that of the 28 S rRNA band (Fig. 1). The OD260/280 was determined by ultraviolet spectrophotometry to be 1.92, indicating that the extracted total RNA sample was of good quality and was not contaminated by degraded DNA, and could be used for subsequent experiment.
Cloning of full-length cDNA of FaPHYC gene from tall fescue
According to the known sequence spliced according to sequencing of the transcriptome of tall fescue in the early stage in laboratory, specific primers for PCR amplification were designed. With the cDNA of tall fescue as a template, PCR was performed, giving a gene fragment of about 600 bp. The PCR product was recovered, and ligated to T vector and sequenced, and an intermediate fragment of PHYC gene of tall fescue with a size of about 644 bp was obtained. The RACE-specific primers were designed using this sequence, and a 192 bp fragment of the 5′ end and a 213 bp fragment of 3′ end were obtained by 5′ RACE and 3′ RACE amplification, respectively. The full-length cDNA of PHYC gene of tall fescue was obtained after splicing. It was 3 816 bp in size (Fig. 3). The result of DNAMAN software analysis showed that the gene consisted of a 213 bp 3′ non-coding region, a 192 bp 5′ non-coding region, and an open reading frame of 3 411 bp in length, encoding 1 136 amino acid residues. It was designated FaPHYC. The BLAST online software (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to predict the conserved domains of the FaPHYC gene, which was found to have two conserved domains. The N terminus was composed of the GAF and phytochrome domains, and the C terminal included two repeated PAS domains, one histidine kinase A domain, and one histidine kinase-like ATPase domain.
Prediction of properties of the protein encoded by FaPHYC gene of tall fescue
The prediction was performed with the online software ProtParam. The results showed that the protein formula of FaPHYC gene was C5556H8870N1538O1673S59, which had the total number of atoms of 17 696 and the molecular weight of about 125 874.39 kDa. Its average hydrophilicity index, isoelectric point pI and instability coefficient were -0.147, 5.81 and 49.54, respectively. This protein was a hydrophilic protein (Fig. 5).
Characteristics of the protein encoded by FaPHYC gene of tall fescue
The structure of FaPHYC protein was analyzed by SOPMA online software. The results showed that α-helix, β-turn, random coil and extended strand of the secondary structure of the target protein accounted for 48.06%, 5.11%, 32.92% and 13.91%, respectively. The results of PSIPRED analysis showed that there were 35 α-helices and 35 β-sheets, and random coils connected α-helices with β-sheets (Fig. 6), which is consistent with the results of SOPMA analysis. The tertiary structure predicted by SWISS-MODEL software was identical to the secondary structure predicted by SOPMA and PSIPRED analysis (Fig. 7). The analysis results of the online software SignalP 4.1 indicated that the protein had no signal peptide sequence.
Analysis of FaPHYC gene expression in tall fescue
Real-time fluorescence quantitative PCR was used to analyze the expression of FaPHYC gene in leaves of tall fescue within 48 h day and night. The results showed that in the first photoperiod under light condition from 20:30 to 0:30, the level continued to increase, reaching the maximum value, while the expression level in the dark begun to decrease gradually, and decreased to the lowest at 04:30; and in the second photoperiod, the expression level continued to rise from 20:30 to 0:30 until the second peak, and then declined continuously. The highest peak of the FaPHYC gene expression can be detected in both photoperiods, and the peak of the first photoperiod was about 2.0 times of that of the second one. It indicated that the expression of FaPHYC gene was regulated by photoperiod, suggesting that this gene may play a certain role in the flowering pathway （Fig. 8）. Conclusions and Discussion
Studies in the model plant Arabidopsis thaliana indicate that the regulation of higher plant flowering is accomplished by the light signal transduction pathway and the genes involved in the circadian clock pathway such as GI (GIGANTEA), CO (CONSTANS) and FT (FLOWERING LOCUS T)［16-18］. The use of phytochrome genes to improve crops can reduce the degree of shade avoidance of crops and increase the density of crops to increase yield quickly and effectively, which has been confirmed in rice and potato research［19-20］. In addition, the maturity of crops can be changed to adapt to seasonal changes in different regions, and the sensitivity of crops to photoperiod can be reduced, thereby increasing the promotion area of short-day crops and long-day crops.
In recent years, the research on PHYC genes regulating photoperiod in plants has progressed rapidly, but there are still many scientific problems to be further explored. In this study, we analyzed the expression of FaPHYC gene and found that the highest peak of FaPHYC gene was detected in both photoperiods and the peak of the transcription level in the first photoperiod was greater than that in the second photoperiod, indicating that the gene is regulated by day and night illumination and affects plant flowering, which accords with the results of Kobayashi et al.［21］. Tall fescue ‘Qiancao No.1’ was bed by Wang et al.［22］from local wild tall fescue plants. It is planted in the short-day sunshine area of Dushan, Guizhou, and blooms for about 240 d, showing late flowering traits. In the long-day sunshine area, Altai, Xinjiang, it is an early-flowering phenotype.
In this study, the full-length cDNA of FaPHYC gene was obtained from tall fescue by 3′ RACE and 5′ RACE technology using tall fescue ‘Qiancao No.1’. It was found by amino acid alignment that this gene had higher similarity with Lolium perenne L., Aegilops tauschii Coss. and Brachypodium distachyon (L.) Beauv., which accords with the fact that they all belong to Gramineae. Protein domain and functional domain analysis indicated that the gene had two conserved domains, and its expression level was further analyzed. As for how the FaPHYC gene in tall fescue is regulated by light under different photoperiod conditions, it is necessary to further construct a vector to transform A. thaliana for functional verification.
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