Material culture
Seeds of decaploid E. elongata were collected from the National Experiment Station of Precision Agriculture, Xiao Tang Shan, China, located approximately 55 km from Beijing (39°34′ N, 116°28′ E). Plump seeds were sterilized with sodium hypochlorite (NaClO) solution (5%, v/v) for 5 min, rinsed thoroughly with distilled water, incubated in 40°C water for 56 h, and then germinated on moistened filter paper for four days at 25°C in the dark. After plumule emergence, uniform seedlings were transferred to plastic containers (20 cm long, 10 cm wide, and 7 cm high) containing modified Hoagland’s nutrient solution [2 mmol·L-1 KNO3, 0.5 mmol·L-1 NH4H2PO4, 0.25 mmol·L-1 MgSO4·7H2O, 1.5 mmol·L-1 Ca(NO3)2·4H2O, 0.5 mmol·L-1 Fe-citrate, 92 μmol·L-1 H3BO3, 18 μmol·L-1 MnCl2·4H2O, 1.6 μmol·L-1 ZnSO4·7H2O, 0.6 μmol·L-1 CuSO4·5H2O, 0.7 μmol·L-1 (NH4)6Mo7O24·4H2O] for four weeks. The nutrient solution was renewed every two days. All seedlings were grown in the same chamber under a day/night cycle of 16 h/8 h at 25°C/18°C, relative humidity of 60%–80%, and a flux density of 600 µmol·m-2·s-1.
Cloning of EeSKOR
Four-week-old E. elongata seedlings were treated with 200 mmol·L-1 NaCl for 24 h. After treatment, fresh roots (200 mg) were washed in sterile water and then ground in liquid nitrogen. Total RNA was extracted with a Trizol Kit (Sangon Biotech. Co., LTD, Shanghai, China) according to the manufacturer’s instructions. Primers EeSKOR-1/EeSKOR-2 were designed according to the principle of high homology and degeneracy by comparing SKOR gene sequences of other plants in the GenBank database (Table S1). The conserved core fragments of EeSKOR were amplified using PCR. The PCR amplification was calibrated at 94°C for 2 min; 30 cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 1 min, and a final extension at 72°C for 10 min. PCR products were purified from agarose gels, ligated into the PMD19-t vector, and sequenced by Sangon Biotech Co, Ltd., (Shanghai, China). The 5'- and 3'- RACE primers were designed by referring to the core sequence of the EeSKOR gene and were obtained according to the corresponding methods of the RACE Kit (Table S1). The gene fragments of 5'- and 3'- RACE were amplified using the Takara Premix PrimeSTAR HS Kit (Takara Biotech Co., Ltd., Dalian, China) according to the manufacturer’s instructions, and the cDNA of EeSKOR gene was spliced and detected by 1.2% agarose gel electrophoresis. The recovered products were connected to the PMD19-t vector, then transformed and cloned, and the positive strain was identified and sequenced by Sangon Biotech.
DNA sequence and phylogenetic analyses
The EeSKOR sequence was analyzed, and the coding regions were predicted using ESPript 3 (Easy Sequencing in PostScript 3) software. EeSKOR sequence homology analysis and phylogenetic tree construction were performed using DNAMAN 6.0 software (Lynnon Biosoft, San Ramon, CA, USA). The isoelectric point and molecular mass were predicted using the online Compute pI/Mw tool (http://web.expasy.org/compute_pi/).
Expression analysis of EeSKOR
Real-Time Quantitative Reverse Transcription methods PCR were used to analyze the expression patterns of the EeSKOR gene in the root, sheath, and leaf of E. elongata subjected to different NaCl concentrations for 24 h (0, 25, 50, 100, 150, and 200 mmol·L-1) and different osmotic potentials of sorbitol for 24 h (0, -0.5, -1.0, and -1.5 MPa). Under each treatment, the extraction of total RNA in the root, sheath, and leaf of E. elongata was carried out according to the RNA Extraction Kit instructions of Takara Biotech. The first strand of cDNA was synthesized according to the instructions for the Prime Script™ RT reagent Kit with gDNA Eraser (Takara Biotech., Co., Ltd.). The qRT-PCR forward primer for the EeSKOR gene was P1 (5'-TACGGAGGCTGCTCAGGTTT-3'), and the reverse primer was P2 (5'-CGCATCTCCTCGCTTCATC-3'); the PCR product length was 189 bp. The positive primer for qRT-PCR of the internal gene Actin was P3 (5'-CTTGACTATGAACAAGAGCTGGAAA-3') and the reverse primer was P4 (5'-TGAAAGATGGCTGGAAAAGGA-3'); the PCR product length was 139 bp. The qRT-PCR experiment was conducted using a StepOnePlus instrument (Thermo Fisher Scientific, Waltham, MA, USA). The reaction system consisted of SYBR® Premix Ex Taq II (Takara Biotech., Co., Ltd.) 12.5 L, both positive and reverse primers 1 L, cDNA 2 L, and water was added to 25 L. The PCR amplification procedures were as follows: denaturation at 95°C for 1 min, 95°C for 5 s, 60°C for 30 s, and 40 cycles. The relative expression of EeSKOR was calculated using the 2-∆∆CT method. Experiments were repeated at least five times to obtain similar results.
Subcellular localization analysis
The positive primers with the Kpn I enzyme loci (5'- GGGGACGAGCTCGGTACCATGGAGAGGGAGATTGTAGCAGAGT-3') and reverse primers with the Xba I enzyme loci (5'- CATGGTGTCGACTCTAGACTGATCGGCTGCAACAGCAGCTGTA-3') were designed using Primer 5.0 software (Premier Biosoft International, Palo Alto, CA, USA). The product templates were EeSKOR open reading frame bacterial liquid that had been sequenced and verified. The target fragments were amplified using the Takara Premix PrimeSTAR HS Kit, and the PCR reaction conditions were 95°C for 5 min, 94°C for 30 s, 58°C for 30 s, 72°C for 1 min, 30 cycles, and a final extension at 72°C for 10 min. The pCAM35-GFP plasmid was digested using KpnI and XbaI, and the results were detected by agarose gel electrophoresis after enzyme digestion. The recombinant plasmid pCAM35-EeSKOR-GFP was obtained by ligating, transforming, and identification of the target fragment and the linearized pCAM35-GFP enzymatic digestion product using the BioTeke DNA purification recovery Kit (BioTeke Corporation, Wuxi, Jiangsu, China). The empty vectors pCAM35-GFP and pCAM35-EeSKOR-GFP were imported into Agrobacterium GV3101 using the freeze-thaw method for future use. The pCAM35-GFP and pCAM35-EeSKOR-GFP were transferred into the epidermal cells of Nicotiana benthamiana by injection, and the expression location of EeSKOR in the epidermal cells of tobacco leaves was observed using a Leica TCS SP8 confocal microscope (Leica Camera AG, Wetzler, Germany) in Germany.
Construction of plant expression vector and transformation of agrobacterium
Nco I and Bgl II restriction sites were introduced at both ends of the upper and lower primers of the EeSKOR open reading frame (ORF), and the target fragment PCR products were detected by 1.2% gel electrophoresis after RT-PCR amplification. The restriction sites on pCAMBIA1301 were double-digested with Nco I/Bgl II, and the large fragments were recovered. Then, the target EeSKOR gene was inserted into the linear plant expression vector (pCAMBIA1301-35S-EeSKOR-Nos) using Clontech In-Fusion seamless connection technology (Takara Biotech Co., Ltd.) according to the manufacturer’s instructions, and the specific bands were obtained by double-digestion with Hind III/Bgl II. The constructed overexpression vector was transformed into the sensing state of Agrobacterium GV3101 by the freeze-thaw method, and the transformed bacterial solution was uniformly coated onto YEP solid medium (50 mg·L-1 kanamycin, 50 mg·L-1 rifampicin, 50 mg·L-1 gentamycin). After inverted culture at 28°C away from light, a single colony was grown after 2–3 days. The single colony was picked up using an inoculation ring, inoculated into 5 mL YEP medium, and incubated overnight. The plasmid was extracted and confirmed as a positive clone by PCR amplification.
Genetic transformation and molecular characterization
The above-mentioned constructs were introduced into Agrobacterium tumefaciens strain GV3101 by the chemical method and then used for tobacco (Nicotiana tabacum cv. Wisconsin 38) transformation using the leaf disc method as described by Horsch et al.32 with minor modifications. The infected leaf sections were cultivated on Murashige and Skoog (MS) medium containing 2 mg·L-1 6-benzylaminopurine (6-BA) and 0.2 mg L-1 1-naphthaleneacetic acid (NAA) at pH 5.8–6.0 for three days. The WT tobacco leaf DNA and agrobacterium-positive bacterial liquid were used as controls. Kan-resistant tobacco leaf DNA was used as a template, EeSKOR-F/EeSKOR-R and DPF1/DPR1 were used as primers (Table S1), and the transgenic tobacco positive plants were detected by PCR amplification. For western blotting, 20 μg tonoplast proteins were separated using 12% (m/v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Blots were performed according to the method described by Kumari et al.33.
Analysis of salt tolerance of transgenic tobacco plants
T1-generation seeds of wild-type tobacco (N. tabacum cv. Wisconsin 38), null vector (Vector), and overexpressed EeSKOR transgenic tobacco (L12 and L36) plants were sterilized with 5% (v/v) sodium hypochlorite (NaClO) solution for 5 min and then washed several times in distilled water. The moist seeds were evenly seeded in a petri dish covered with absorbent paper, and dark cultured at 25°C for four days. After germination and rooting, the seeds were transferred into a black culture box (20 cm×10 cm×7 cm) and seeded with Hoagland nutrient solution. Light culture was conducted for 16 h (day)/8 h (night) at a light intensity of about 600 mol·m-2·s-1 with the Hoagland nutrient solution replaced after two days; air relative humidity was kept at 60%–80%. The WT, Vector, and transgenic tobacco (L12 and L36) plants were subjected to twenty-one days of stress in Hoagland nutrient solution with NaCl concentrations of 0, 50, 100, 150, and 200 mmol·L-1, respectively. The plant height, biomass, H2O2, MDA, and chlorophyll (Chl) content, SOD activity, and Na+ and K+ accumulation concentrations in the aboveground part and root of the plant were measured. After fresh weight measurements, roots, sheaths, and leaves were oven-dried at 80°C to a constant weight, and the dry weight of each organ was recorded. The MDA and Chl contents were determined using the method of Zhou34, and the SOD activity was determined by the nitroblue tetrazolium (NBT) method. Na+ and K+ concentrations in the roots, sheaths, and leaves were measured using a flame emission spectrophotometer.
Statistical analysis
Each treatment was repeated five times independently with three seedlings per replicate. All data are presented as mean ± standard deviation (SD). Duncan’s multiple range tests were performed using statistical software (v.13.0, SPSS Inc, Chicago, IL, USA).