Cloning and expression analyses of a Pyrabactin Resistance 1 (PYR1) gene from Magnolia sieboldii K. Koch

ABSTRACT Magnolia sieboldii K. Koch is endemic to China and has high medicinal and ornamental values. However, its seed exhibits morphophysiological dormancy, and the molecular mechanisms of which are not clearly understood. To reveal the regulation mechanism of the ABA signal in seed dormancy, the M. sieboldii ABA receptor Pyrabactin Resistance 1 (PYR1) gene was cloned and analyzed. Analysis of the MsPYR1 sequence analysis showed that the full-length cDNA contained a complete open reading frame of 987 bp and encoded a predicted protein of 204 amino acid residues. The protein had a relative molecular weight of 22.661 kDa and theoretical isoelectric point of 5.01. The transcript levels of MsPYR1 were immediately upregulated at 16 DAI and then decreased at 40 DAI. The highest transcript level of MsPYR1 was found in the dry seeds, indicating that the MsPYR1 gene may play an important role in the regulation of dormancy. The MsPYR1 gene cDNA was successfully expressed in E. coli Rosetta (DE3), and the protein bands were consistent with the prediction. The Anti-MsPYR1antibody could detect the expression of MsPYR1 in M. sieboldii. The results provided a foundation for further study of the function of the MsPYR1 gene. ABBREVIATIONS ABA: Abscisic acid; MPD: morphophysiological; PYR1: Pyrabactin Resistance1; PYL: Pyr1-Like; RCAR: Regulatory Components of Aba Receptors; PP2C: protein phosphatases 2C; SnRK2: sucrose non-fermenting1-related protein kinase2; DAI: day after imbibition; NCBI: National Center for Biotechnology Information; BCA: Bicinchoninic acid; CDD: Conserved Domains.


Introduction
Magnolia sieboldii K. Koch is widely distributed in China, Korea, and Japan. M. sieboldii has high ornamental and medicinal values [1]. The plant had been designated as the City Flower of Benxi in P.R. China and the National Flower of North Korea. Given the difficulty of natural regeneration, the population of M. sieboldii has been decreasing. M. sieboldii has been classified in the IUCN Red List because of 'least concern' [2]. The seed dormancy type belongs to the levels of morphophysiological (MPD) [3]. Abscisic acid (ABA) can affect seed germination by modulating ABArelated functional genes. However, the molecular mechanism of the MPD of the M. sieboldii seed remains unknown.
The phytohormone ABA regulates plant growth and development processes, such as seed dormancy, fruit ripening, leaf senescence, and stress resistance [4]. ABA signaling is required for vegetative and reproductive growth, stomatal aperture, and transcriptional response to the hormone [5]. The ABA signal transduction cascade is one of the best characterized input transmission pathways of plants at the molecular level. The ABA signaling pathway has identified a large number of proteins [6]. Pyrabactin Resistance1 (PYR1)/Pyr1-Like (PYL)/ Regulatory Components of Aba Receptors (RCAR) ABA receptors, clade A type protein phosphatases 2 C (PP2C) and group III sucrose non-fermenting1-related protein kinase2 (SnRK2) subfamily are essential core components of the upstream signal transduction network that regulates ABA-responsive processes, including dormancy and germination [7][8][9][10].
The PYR/PYL/RCAR ABA receptor specifically perceives ABA. The PYR/PYL/RCAR ABA receptors have identified 15 members in the tomato [11] and 14 members in Arabidopsis [5]. PYR1 is critical for seed germination and seedling establishment [10], positive regulation of fruit ripening [12], reducing vegetative growth and seed production [5], inducing stomatal closing movement [13], and response to dehydration stress [14]. The expression of PYR1 in the seeds is higher than that in the other tissues [15]. PYR1 is necessary for ABA signaling in vivo. A study has shown that ABA binds to PYR1 and inhibits PP2Cs [15]. The primary objective of the present study was to clone the PYR1 gene from M. sieboldii, study its expression at different germination stages. An attempt was made to express MsPYR1 protein. The cloning, transcription, and translation analysis of the PYR1 gene provide a foundation for further study on the molecular mechanism of the ABA signal.

Plant material and germination assays
M. sieboldii trees were grown at the Shenyang Botanical Garden (41° 850 N, 123° 727 E, 28 masl) in Shenyang, Liaoning, China. Fresh matured seeds were collected from 20 trees with average height of 7 m on 20 October 2018. The coat was removed, and the seeds were surface sterilized using potassium permanganate (0.05% [w/w]) for 15 min and rinsed thrice with sterilized distilled water. The seeds were allowed to imbibe different treatments, i.e., 10 mM ABA and water, in Petri dishes at 20°C for 48 h. Each treatment had three biological replicates of 100 seeds per Petri dish. After imbibition, the seeds were cold stratified in the dark at 4°C. Seed samples were collected at 0 h imbibition (dry seed, GZ), 0 day after imbibition (DAI) and every 8th DAI then stored at −80°C for RNA extraction.

RNA extraction and cDNA synthesis
The five key time points for seed germination were as follows: dry seed, 0 DAI, 16 DAI, 40 DAI, and 56 DAI. The seeds were collected at these time points and used for total RNA extraction. The RNA was extracted using the RNA Simple Total RNA Kit (Tiangen, Beijing, China) [16]. The concentration and purity of the RNA obtained were detected using Nanodrop2000 UV electrophotometer (Thermo Scientific). The cDNA was synthesized using the GoScript TM Reverse Transcription System (Promega).

PYR1 gene cloning and sequencing
The full length of the primers of PYR1 (Table 1) was designed based on the transcriptome data by using the Primer 5 software (unpublished). The PYR1 clone was amplified using PrimeSTAR® HS DNA Polymerase (TaKaRa). RT-PCR reaction system was used in a 50 μL of reaction mixture including PrimeSTAR HS Taq (25 μL), primers (2 μL), cDNA template (1 μL), and ddH 2 O. The RT-PCR reaction conditions were as follows: 30 s at 98°C; 30 cycles of 10 s at 98°C, 15 s at 58°C, 1 min at 72°C; and a 10 min extension at 72°C. The PCR fragments were connected into the pMD18-T vector (TaKaRa) and sequenced by TaKaRa Co., Ltd.

Quantitative real-time PCR (qRT-PCR)
The qRT-PCR procedure was established using the qTOWER 2.0/2.2 Real Time PCR Systems (USA). All primers are listed in Table 1. The qRT-PCR reaction system was used in a 20 μL reaction mixture including PrimeSTAR HS Taq (10 μL), primers (1.6 μL), cDNA template (2 μL), and ddH 2 O (6.4 μL). The qRT-PCR reaction conditions were as follows: 30 s at 95°C; and 40 cycles of 5 s at 95°C, 20 s at 60°C. The data were analyzed using the 2 −ΔΔ Ct method described by Zhang et al [17].

Prokaryotic expression of the PYR1 gene
The prokaryotic expression vector pET28a-PYR1 was constructed by PCR with pMD18-T-PYR1 as template. The pET28a-PYR1 construct was transformed into E. coli Rosetta (DE3). The positive clones were screened by PCR with pET28a-F and pET28a-R as primers ( Table 1). The monoclones were selected and inoculated in fresh LB liquid medium (containing 50 mg/L kanamycin) for approximately 4 h at 37°C, and the OD 600 was approximately 0.6. IPTG with a final concentration of 0.5 mM was added to induce the expression of PYR1 [19].

Purification of the recombinant protein
The bacterial culture solution was induced at 15°C for 15 h, and then the lysed cell was ultrasonicated. The cell lysates were loaded to the chromatographic columns with Ni IDA resin. The column was washed with 200 mL of the wash buffer containing 2 M NaCl and 15 mL of the wash buffer containing 50 mM NaCl. The proteins were eluted with 20 mL of an elute buffer containing 20 mM iminazole, then the proteins were eluted with 20 mL of another elute buffer containing 250 mM iminazole [20]. Approximately 1 mg/mL of pure recombinant PYR1 was obtained.

Antibody preparation and western blot analyses
After the concentration of the Bicinchoninic acid (BCA) protein was determined, two New Zealand rabbits (2-2.5 kg) were injected subcutaneously with 400 µg, immunized once at 1.5 weeks and the process was performed four times. Blood samples were collected for detection, and the titer of the antiserum against PYR1 was determined by indirect ELISA. The titer was greater than 1:500,000, and the final blood samples were collected to prepare the antiserum. Western blot analysis was performed as described [21]. After 12% SDS-PAGE electrophoresis, the gels were transferred onto a Trans-BlotTurboTM (BioRad) transfer system. Anti-PYR1 antibody (1:500) was used as the primary antibody and anti-rabbit IgG (1:5000) as the secondary antibody.

Cloning and sequence analysis of MsPYR1
The full-length PYR1 cDNA was obtained from M. sieboldii seed by RT-PCR. The length of the PCR product contained a complete ORF of 615 bp and encoded a predicted protein of 204 amino acid residues ( Figure 1). The cDNA sequence was submitted to GenBank under the accession number MW505908.
Multiple amino-acid sequence alignment revealed homology between the predicted aminoacid sequence of MsPYR1 and other PYR1 proteins ( Figure 2 To evaluate the molecular evolutionary relationships among MsPYR1 and other PYR1s, the phylogenic tree was constructed using the amino acid sequence through the MEGA7.0 software. MsPYR1 was more closely related to Arabidopsis thaliana, Vitis vinifera, and especially Cinnamomum micranthum (Figure 3).

Bioinformatic analysis of MsPYR1
The relative molecular weight of the MsPYR1 protein was 22.661 kDa, and the theoretical isoelectric  point of the protein was 5.01. The amino acid with the highest content in the protein was Glu (9.8%), and the lowest content was Trp (0.5%) ( Table 2).
The conserved domain of the MsPYR1 protein was predicted by Conserved Domains (CDD) in the NCBI. The MsPYR1 protein had a structural domain of the PYR/PYL/RACR protein family, which in turn belonged to the START/ RHO_alpha_C/PITP/Bet_v1/CoxG/CalC superfamily and contained putative hydrophobic ligand binding sites, protein interfaces, and gate structures ( Figure 2). The SMART analysis showed a Polyketide_cyc2 domain Leu 53th to Glu 194th (Figure 1). The three-dimensional structure of the MsPYR1 protein was predicted using Normal model and the crystal structure of FePYR1 was selected as a template, the sequence identity between MsPYR1 protein and FePYR1 protein was 88% (Figure 4).

Transcription analysis of MsPYR1 in the different stratification stages
qRT-PCR was used to analyze the transcription of MsPYR1 at different stratification stages ( Figure 5). The highest MsPYR1 transcript was detected in the dry seeds. During stratification, the PYR1 transcript was immediately upregulated at 16 DAI and then decreased. The lowest expression was observed at 40 DAI, during which the seed coat began to crack. Therefore, MsPYR1 may play a vital role in the imbibition and seed germination. In Arabidopsis, PYR/PYLs were also expressed at high levels in the dry seeds, which   were more sensitive to ABA [15]. PYR/PYLs play a crucial role in the regulation of seed dormancy and stomatal switch. Compared with the wild type, the pyr/pyls mutants were less sensitive to ABA [22,23]. This characteristic could explain the beginning of the cracking of the seed coat and the low expression level of MsPYR1 at 40 DAI. So, ABA is a major plant hormone involved seed germination and early seedling development [24,25].

Prokaryotic expression of MsPYR1 and immunoblot analysis
The recombinant plasmid PET28A-MSPYR1 was transformed into the E. coli Rosetta (DE3) cell.
After induction with 0.5 mM IPTG at 37°C for 2 h, 12% SDS-PAGE electrophoresis was performed ( Figure 6). The results showed that after IPTG induction, a protein band appeared near 26 kDa, which was slightly larger than the theoretical molecular weight of 22.661 kDa. This characteristic may be affected by the his-tag. After expansion, the MsPYR1 protein was purified by using 6× His protein purification kit. E. coli Rosetta (DE3) is frequently used for the high expression of eukaryotic or prokaryotic proteins [26,27]. The protein was loaded into a Ni-IDA affinity column to bind the polyhistidine-tagged recombinant MsPYR1. The purity of MsPYR1 eluted from the first Ni-IDA affinity column with 20 mM imidazole was approximately 20%. After purification  of the second Ni-IDA affinity column with 250 mM imidazole, MsPYR1 was approximately 80%, which was exhibited as a single band on the SDS-PAGE with the molecular weight of 26 kDa ( Figure 6). The expression product easily formed inclusion bodies, which increased the difficulty for protein purification [28]. To avoid the formation of inclusions and enhance the soluble yield of the target protein, the expression was performed at a low temperature (15°C) [29,30]. In this study, the E. coli Rosetta (DE3) strain had a low expression level of the recombinant proteins as inclusion bodies (Figure 7).
Anti-MsPYR1 antibodies are used as a convenient detection tool to bind/recognize the target proteins with higher stability and affinity. The presence of a cross-reacting protein appeared at the approximate size of 26 kDa in the purification of recombinant proteins and seed protein during the immunoblot analysis ( Figure 8). When the primary antibody was diluted 500 times, the recombinant protein (2 ng) and seed protein 100 (µg) could be detected by the assay.

Conclusions
In the present study, an ABA receptor gene, MsPYR1 was cloned and successfully expressed in E. coli. The expression pattern of MsPYR1 at different stratification stages was analyzed using qPCR. The highest transcript level of MsPYR1 was found in the dry seeds. The pure recombinant MsPYR1 protein was successfully obtained. The Anti-MsPYR1 antibody could detect the expression of MsPYR1 in M. sieboldii. The results provided a foundation for further study of the function of the MsPYR1 gene. The results of present study provide valuable information for further functional studies of MsPYR1 gene.