Cloning and Characterization of Phytochrome A Gene FaPHYA from Tall Fescue

Xiaoli WANG, Jiahai WU, Jianhong SHU, Yiming CAI, Xiaoxia LIU, Xiaodong LI

Guizhou Institute of Prataculture, Guiyang 550006, China

Light is one of the important environmental factors affecting the growth and development of plants,and it is also an external development signal regulating photomorphogenesis in plants. Studies have shown that light interacts with the development processed in plants through photoreceptors,regulating the expression of associated genes[1-2].Currently,three main types of photoreceptors have been identified in plants, including phytochrome, cryptochrome and phototropin. Among them, phytochrome is a class of photoreceptor that is studied most deeply[3]. They are a more complex gene family, and play an important role in the regulation of photomorphogenesis in plants. In plants,the PHY family includes at least five members (PHYA, PHYB, PHYC,PHYD, PHYE) which perform different physiological functions. Among them,PHYA and PHYB are studied most deeply[4]. Phytochrome senses light signals, and it participates in a variety of physiological activities, such as seed germination, seedling de-etiolation, geotropism, phototropism, photofobotaxis,leaf unfolding,stem elongation, chloroplastic activities, stomatal opening,flowering and crop yielding[5-7].In addition, resent studies have found that the phytochrome is also involved in the regulation of resistances in plants under biotic and abiotic stresses,such as the resistances to pathogens[8-13],pests[14-19],cold injury[20-21],high temperature[22-25], drought[26-31]and salt injury[32-34].

Tall fescue is a Festuca plant,and it is widely used in city landscaping and water and soil conservation due to its rapid growth, disease resistance and wide adaptability. Moreover, tall fescue has become an ideal material for mining stress-resistant genes from plants[35-36]. In view of this, a phy tochrome A gene was isolated from tall fescue,and its bioinformatics and taxonomic status were studied so as to lay a foundation for improving pasture resistance though genetic modification of associated genes in phytochrome signaling pathway in gramineous pasture production.

Materials and Methods

Materials

Plant material Qiancao 1 is new national forage cultivar bred by the Guizhou Institute of Prataculture in 2005.Its registration number is 299.

Main reagents The E. coli DH5α competent cells were purchased from the Tiangen Biotech (Beijing) Co., Ltd.The cloning vector pMD 19-T, RNA extraction kit (TaKaRa RNAiso Reagent), RNA reverse transcription kit (RevertAid First cDNA Synthesis Kit) were purchased from the Thermo Scientific Company. The 5’RACE kit(5’RACE System for Rapid Amplification of cDNA Ends, Version 2.0) was purchased from the Shanghai Invitrogen Biotechnology Co., Ltd. The3’RACE kit (SMARTerTMRACE cDNA Amplification Kit) was purchased from the Clontech Laboratories Inc. The DNA Marker DL2000, 100 bp DNA Ladder Marker, LA Taq enzyme,EASY Dilution, DNaseI and In Vitro Transcription T7 kit were purchased from the TaKaRa Biotechnology(Dalian) Co., Ltd. The Taq enzyme(Advantage 2 PCR kit)was purchased from the Clontech Laboratories Inc.The agarose DNA extraction kit was purchased from the Omega BioTek Inc.The yeast extract,peptone and agarose were purchased from the Oxoid Inc.The X-gal,IPTG and Ampicillin were purchased from the Beijing Soledad Technology Co.,Ltd.

Table 1 Sequences of primers for amplifying cDNA sequence of FaPHYA gene in tall fescue

Methods

Total RNA extraction from tall fescue The total RNA in leaves of Qiancao 1 was extracted using extraction kit (TaKaRa RNAiso Reagent).The absorbances of the extracted RNA at 260 and 280 nm were determined with a UV spectrophotometer.The A260/A280ratio was calculated, and the concentration (μg/ml)of total RNA was calculated (A260× 40 × dilution time).The integrity of extract RNA was examined by 1.0% agarose gel electrophoresis. And then, the RNA was preserved at-80 ℃for use.

Primer design The sequence fragment of phytochrome A gene in tall fescue,obtained by transcriptome sequencing was used as a template. To validate the accuracy of the fragment assembly, two pairs of primers were designed using Primer Premier 5.00,and they are named as FaPHYA-fwd1,FaPHYA-rev1, FaPHYA-fwd2 and Fa-PHYA-rev2, respectively. In addition,three specific downstream primers were designed, and they were combined with the primers in the 5’RACE kit to clone the 5’ end of the FaPHYA gene. The designed primers were named as FaPHYA-rev3, FaPHYArev4 and FaPHYA-rev5. Similarly, two specific upstream primers were designed, and they were combined with the primers in the 3’RACE kit to clone the 3’ end. The designed primers were named as FaPHYA-fwd3 and Fa-PHYA-fwd4(Table 1).

Full-length cDNA cloning of Fa-PHYA gene in tall fescue Combing the designed primers and provided primers by the 3’RACE and 5’RACE kits, the 5’ and 3’ ends of the phytochrome A gene in tall fescue were cloned as described by the instructions.The recovered PCR product was inserted in the T vector and cloned.The amplified fragment was sequenced and then spliced with known sequences by DNAMAN. Thus the full-length cDNA sequence of phytochrome A gene in tall fescue was obtained.

Functional analysis of protein encoded by FaPHYA gene in tall fescue The amino acid sequences were spliced using DNAMAN. Based on amino acid sequences, the homology analysis was performed for FaPHYA from tall fescue and PHYAs from other plants.The open reading frame of Fa-PHYA gene was searched in NCBI,and the functional domains of the protein encoded by FaPHYA were analyzed (http://www.ncbi.nlm.nih.gov/gorf/gorf.html; http://www.ncbi.nlm.nih.gov/structure/c dd/wrpsb. cgi). The relative molecular weight, isoelectric point and hydrophobicity of FaPHYA were analyzed using ExPASy (http://cn.expasy.org/tools/protparam. Html;http://www.expasy.org/cgi-bin/protscale.pl). The secondary structure of Fa-PHYA was analyzed using SOPMA(http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl). The subcellular localization analysis was performed at http://wolfpsort.seq.cbrc.jp/. The new accession number was applied in GenBank.

Results and Analysis

Cloning and nucleic acid sequence analysis of FaPHYA gene in tall fescue

Using the sequence fragment of PHYA in tall fescue, obtained by transcriptome sequencing, as a template,the accuracy of splicing was validated with the amplified region-overlapped primers. The extracted total RNA from tall fescue was transcribed into cDNA.Using the cDNA as a template, the RT-PCR was performed with primers FaPHYA-fwd1, FaPHYA-rev1, Fa-PHYA-fwd2 and FaPHYA-rev2. As shown in Fig.1 and Fig.2,two apparent bands in size of 1 140 and 1 397 bp were obtained. The amplification products were cloned by inserting into T-vector and sequenced. The sequence fragments were spliced and aligned with the sequence of PHYA obtained by transcriptome sequencing.It was found that a fragment, in length of 198 bp, was deleted in the sequence of PHYA obtained by transcriptome sequencing. Subsequently,the verified PHYA sequence was used as a template, and PCR was performed by combing the designed specific primers and provided primers by the 3’RACE and 5’RACE kits. Thus the 3’ and 5’ ends of the PHYA gene were cloned. The amplification products,in size of 1 062 and 905 bp were obtained (Fig.3, Fig.4). The sequencing results showed that there were overlapping regions between the cloned fragments and known sequences, indicating that the cloned fragments were the 5’ and 3’ ends of mRNA sequence of PHYA gene in tall fescue. The three sequences were spliced using DNAMAN, thus the fulllength cDNA sequence (3 983 bp) of PHYA gene in tall fescue was obtained. It was named as FaPHYA with accession number of KP202858 in GenBank.

Functional analysis of protein encoded by FaPHYA gene in tall fescue

The cDNA sequence of FaPHYA gene in tall fescue was analyzed using ORF Finder in NCBI, and one complete open reading frame was found.Its initiation codon is located at the 293thnucleotide, while stop codon is located at the 3 682thnucleotide(Fig.5). So it was speculated that the encoded protein by the FaPHYA gene is composed of 1 129 amino acids(Fig.6).The molecular weight and isoelectric point of FaPHYA were speculated as 125.23 kDa and 5.73,respectively. The subcellular localization analysis showed that the protein is located in the cytoplasm. The hydrophobicity of FaPHYA protein was analyzed using ExPASy’s ProtScale.The results showed that the maximum hydrophobic value is 2.978,while minimum value is-3.244.Below the score of -2, there are 12 hydrophilic peaks(Fig.7).The secondary structure of the FaPHYA protein was predicted using SOPMA.The protein contains approximately 46.68% of α-helix, 14.61% of extended strand, 5.76% of β-corner and 32.95%of irregular curling(Fig.8).

Conserved domain analysis off Fa-PHYA

The phytochrome PHYA is normally composed of two parts, N- and C- terminals. The N- terminal is a photosensitive area, and the C- terminal is a regulatory region. The domain analysis of FaPHYA (Fig.9) showed that the N- terminal is composed of GAF (217-401 AA) and Phytochrome(413-593 AA) domains, and the C-terminal contains two repeated FAS domains (629-735 AA,762-873 AA),one histidine kinase A domain (895-951 AA) and one histidine kinase-like ATPase domain (1 014-1 117 AA).Based on the prediction result, the amino acid sequence of FaPHYA from tall fescue was aligned with those of Brachypodium distachyon, Arabidopsis thaliana, Oryza sativa, Avena sativa L and Zea mays. The results showed that the amino acid sequence of FaPHYA contains one GAF domain,one Phytochrome domain, two PAS domains, one histidine kinase A domain and one histidine kinase-like ATPase domain(Fig.10).The domains of FaPHYA protein all have relatively conserved amino acid sequences,suggesting that the FaPHYA in tall fescue may have similar light signaling functions with phytochrome A pro-tein in Arabidopsis thaliana or Oryza sativa.

Homology analysis of FaPHYA gene

In order to study the genetic evolutionary relationships between Fa-PHYA from tall fescue and PHYAs from other plants, the phylogenic tree of PHYAs was constructed based on homology analysis of the amino acid sequences. The homologies among amino acid sequences encoded by PHYA genes can basically reflect the phylogenetic relationships among the species. The phytochrome As of monocots are cluster into one large group, while the phytochrome As of dicots are clustered into one group.The FaPHYA of tall fescue, as a monocot, shows higher homologies(up to 85% ) with PHYAs of other monocots, indicating closer genetic relationships. But the genetic relationships between FaPHYA and PHYAs of dicots are farer (Fig.11). The homologies of amino acid sequences of PHYA proteins between Festuca arundinacea and Avena sativa L,Brachypodium distachyon, Triticum,Oryza sativa and Arabidopsis thaliana are 96.99%,92.04%,89.82%,88.24%and 62.33%,respectively(Fig.11).

Conclusions and Discussion

The predicted amino acid sequence of FaPHYA in tall fescue was aligned with those of PHYAs in Brachypodium distachyon, Arabidopsis thaliana, Oryza sativa, Avena sativa L and Zea mays. The functional domains of FaPHYA all have obvious conserved amino acid sequences. It suggests that as the primary photoreceptor,the FaPHYA in tall fescue may have the similar light signaling functions compared with PHYAs in Arabidopsis thaliana and other gramineous plants. In tall fescue, FaPHYA makes responses to a series of development pathways excited by far-red light. Previous studies show that the phytochrome PHYAs are generally composed of the N-and C-terminals,of which the N-terminal is photoreceptor region and C-terminal is regulation region. After activated by far-red light,PHYAs can form homodimers through the interaction between C-terminals,and only the homodimers can enter the nucleus to activate the expression of downstream genes[7]. In this study,the bioinformatics analysis showed that the N-terminal of FaPHYA is composed of GAF(217-401 AA)and Phytochrome(413-593 AA)domains,and the C-terminal contains two repeated FAS domains(629-735 AA,762-873 AA), one histidine kinase A domain(895 -951 AA) and one histidine kinase-like ATPase domain (1 014-1 117 AA)[37-38]. Moreover, the repeated PAS domains in the C-terminal have the functions of providing platform for protein-protein interaction,regulating small ligands and localizing karyophilic proteins in nuclei[39-40].

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