PeBL1, a novel protein elicitor from Brevibacillus laterosporus

21 In our study, we report the identification, characterization, and gene cloning of a novel 22 protein elicitor (PeBL1) secreted from Brevibacillus laterosporus strain A60. Through a 23 purification process consisting of ion exchange chromatography and HPLC, we isolated a 24 protein that was identified by ESI-QTOF-MS/MS. The 351-bp PeBL1 gene produces a 25 12833 Da protein with 116 amino acids that contains a 30-residue signal peptide. The 26 PeBL1 protein was expressed in Escherichia coli. The recombinant protein can induce a 27 typical hypersensitive response (HR) and systemic resistance in Nicotiana benthamiana 28 as the endogenous protein. PeBL1-treated N. benthamiana exhibited a strong resistance to 29 the infection of TMV-GFP and P. syringae pv. tabaci compared to control N. 30 benthamiana. In addition, PeBL1 triggered a cascade of events that resulted in defense 31 responses in plants, including reactive oxygen species (ROS) production, extracellular 32 medium alkalization, phenolic compounds deposition and several defense-related genes. 33 Real-time quantitative PCR analysis indicated that the known defense-related genes PR-1, 34 PR-5, PDF1.2, NPR1 and PAL were up-regulated to varying degrees by PeBL1. This 35 research not only provides insights into the mechanism by which beneficial bacteria 36 activate plant systemic resistance but also sheds new light on a novel strategy for 37 biocontrol using strain A60. 38 39 40 on O cber 7, 2017 by gest ht://aem .sm .rg/ D ow nladed fom INTRODUCTION 41 In nature, plants live in complicated surroundings with various beneficial microbes and 42 potential plant pathogens. In order to prevent infection by pathogens, plants have evolved 43 defense mechanisms leading to a basic innate immunity (1, 2). Additionally, beneficial 44 bacteria can generate a protective action which indirectly makes plant resist the infection 45 of further pathogens through the elicitation of plant defense system (3). This defensive 46 capacity is systemic, for example, root treatment with beneficial bacteria could extend to 47 aboveground plant parts, triggering resistance in the whole plant. Resistance responses 48 triggered by nonpathogens is so-called induced systemic resistance (ISR), which can 49 efficient resist a broad spectrum of pathogens, including bacteria, fungi, viruses, 50 nematodes, and insects (4-6). ISR is phenotypically similar to the well-studied systemic 51 acquired resistance (SAR) motivated by an incompatible pathogen (7). Among these 52 ISR-inducing bacteria, most are plant growth-promoting bacteria (PGPB), which are 53 related to many plant species and generally present in a variety of environments (8). The 54 most well-studied class of PGPB are plant growth-promoting rhizobacteria (PGPR) 55 colonizing the root surfaces and the rhizosphere (9). 56 ISR has been documented in lots of plant species, for example, Arabidopsis thaliana, 57 tomatoes, beans, cucumbers, radishes and tobacco (2, 6). Globally, ISR has been 58 considered as a three-step procedure that contains perception of the elicitor, signal 59 transduction, and defense-related gene expression triggering the resistance in the whole 60 on O cber 7, 2017 by gest ht://aem .sm .rg/ D ow nladed fom plant. ISR-associated signal transduction mechanisms have been demonstrated, even if 61 comparatively less well understood than SAR. The salicylic acid (SA)-, jasmonic acid 62 (JA)-, and ethylene (ET)-dependent pathways are major players in the regulation of 63 signaling networks that are involved in induced defense responses (10-12). SA is an 64 important signaling molecule involved in the induction of systemic resistance and local 65 defense reactions (13). Several pathogenesis-related (PR) genes, such as PR-1a, PR-1b, 66 PR-5, are generally used as markers of SA-dependent defenses. JA, a signaling molecule, 67 is involved in many different aspects of plant biology including defense and development 68 (14). ET regulates several processes in plants and has been implicated in defense 69 responses. Normally, ISR is mediated by a signaling pathway in which JA and ET play 70 key roles (6, 15). However, Saskia et al. demonstrated that activation of an SA-dependent 71 pathway is a feature of ISR-inducing biocontrol bacteria (16). Maurhofer et al. reported 72 that ISR induced by P. fluorescens strain CHA0 in tobacco is related to PR protein 73 accumulation, suggesting that ISR and SAR share similar mechanisms. Thus, the defense 74 mechanisms of ISR must be further studied (17). 75 The plant resistance system is the condition of enhanced defensive capacity. Plant defense 76 responses triggered by elicitors of biotic and nonbiotic origins are a part of the plant 77 resistance and play important roles in the signal exchange between the plant and the 78 microbe. The elicitors, derived from various organisms including bacteria, fungi, viruses 79 and oomycetes, have different chemical natures and include proteins, glycoproteins, 80 on O cber 7, 2017 by gest ht://aem .sm .rg/ D ow nladed fom peptides, lipids and oligosaccharides (18-20). For example, Harpins are multifunctional 81 protein elicitors produced by Gram-negative plant pathogenic bacteria (21). The fungal 82 elicitors Hrip1, PevD1, and MoHrip1 from Verticillium dahliae, Alternaria tenuissima, 83 and Magnaporthe oryzae, respectively, have been applied to plants to improve their 84 pathogen resistance (22-24). However, a few reports have focused on elicitors that were 85 isolated from biocontrol bacteria. For example, Dimethyl disulfide (DMDS), an elicitor 86 produced by an ISR-eliciting B. cereus strain, can suppress plant fungal diseases and play 87 a crucial role in ISR by B. cereus C1L (25). Massetolide A produced by Pseudomonas 88 fluorescens SS101 is involved in ISR-eliciting defensive capacity in tomato against 89 Phytophthora infestans (26). Surfactins and fengycins produced by Bacillus subtilis S499 90 can also act as elicitors of ISR (5). In contrast to the many researches performed with 91 PAMPs used as models for early defense-related events, very little information is 92 available about the perception mechanisms of ISR-specific protein elicitors (27). 93 In general, a defense reaction triggered by elicitors can be divided into two stages. The 94 first stage occurs minutes after using an elicitor and contains ion fluxes across the cell 95 membrane, extracellular medium alkalization and ROS. In the plant defense reaction, 96 ROS are considered to play an important role in the elicitor signal transduction system 97 and be also associated with the hypersensitive response (28), as a marker of the plant 98 defense reaction (29, 30). ROS have been demonstrated to be sufficient for the induction 99 of plant secondary metabolite accumulation and are required in the plant defense reaction 100 on O cber 7, 2017 by gest ht://aem .sm .rg/ D ow nladed fom (27, 31, 32). The second stage occurs hours after elicitor ingestion and involves the 101 activation of defense-related genes correlated with cytoderm reinforcement, the synthesis 102 of phytoalexins, the accumulation of PR proteins and induction of defense compounds, 103 such as phenolic compounds, callose and PAL (phenylalanineammonia lyase) (33). 104 Brevibacillus laterosporus, a gram-positive, aerobic spore-forming bacterium previously 105 classified as Bacillus laterosporus, can produce diverse metabolites with antifungal 106 activity, which can control the infection of plant pathogens as biocontrol agents (34). We 107 have previously screened a novel strain A60 that was isolated from the soil of mango 108 plants in Changjiang, Hainan province, China (E108 ◦ 46.029’N19 ◦ 15.635’), which was 109 identified as B. laterosporus by phenotypic characterization and 16S rRNA sequencing 110 (35). In addition to antimicrobial activity, strain A60 also exhibited the induction of 111 systemic resistance in numerous types of plants, such as wheat, pepper and Chinese 112 cabbage. The control efficiencies against Phytophthora capsici and Peronospora 113 parasitica in pepper and Chinese cabbage that were treated with strain A60 Aqua (5×10 9 114 Cfu/mL) were 81.6% and 73.7%, respectively, after 10 days. In particular, the yield of 115 Chinese cabbages after treatment with A60 Aqua (5×10 9 Cfu/mL) increased by 13.2% 116 compared to the wild type. Based on the excellent effect, microbial fertilizer of B. 117 laterosporus strain A60 Aqua has been registered (No. 2014-2058) and has achieved 118 large-scale production in the factory of Henan province, with an annual output of 5000 119 tons. The application area has increased to three millions of acres. In previous studies, a 120 on O cber 7, 2017 by gest ht://aem .sm .rg/ D ow nladed fom novel antimicrobial peptide BL-A60 with a molecular mass of 1602.0469 Da was isolated 121 and purified from strain A60 (35). However, the metabolites involved in the activation of 122 systemic resistance by strain A60 have not been completely studied. 123 In this study, we report the purification and characterization of the novel protein elicitor 124 PeBL1 from B. laterosporus strain A60. PeBL1 activated certain early plant defense 125 responses and the systemic resistance in N. benthamiana against infection by TMV-GFP 126 and P. syringae pv. tabaci. Our research helps to elucidate the mechanisms of N. 127 benthamiana systemic resistance triggered by the PeBL1 and provides a novel strategy 128 for using B. laterosporus strain A60 to control plant disease. 129 MATERIALS AND METHODS 130 Bacteria and plant cultivation 131 The strain A60 was preserved at the China General Microbiological Culture Collection 132 Center (CGMCC No:5694) and maintained on Luria-Bertani medium (LB: 10 g tryptone, 133 5 g yeast extract, 10 g NaCl per 1 L distilled water ) at 37 °C in the dark. N. benthamiana 134 seeds were germinated on 1/2 Murashige and Skoog (MS) medium in a growth chamber 135 that was maintained at 25°C with 12 h of light and 12 h of darkness. Following 136 germination, the seedlings were transferred to an autoclaved soil mix containing 1:3 (v/v) 137 high nutrient soil and vermiculite in 8×7.5×7.5-cm pots. One plant per pot was kept in the 138 growth chamber at 25°C with 50% humidity and 16 h of light. 139 Establishment of N.benthamiana cell culture 140 on O cber 7, 2017 by gest ht://aem .sm .rg/ D ow nladed fom Tobacco (N. benthamiana) seeds were soaked in 75% ethanol for 45 sec and in 10% 141 sodium hypochlorite for 10 min, followed by three washes with sterilized water. The 142 sterilized N. benthamiana seeds were cultivated for callus in MS medium. The callus 143 were cut into small pieces after 15 days and suspended in liquid MS medium at pH 5.0 144 supplemented with inositol (100 mg/ml), 0.2% KH2PO4, 3% sucrose, 145 2,4-dichlorophenoxyacetic acid (0.2 mg/ml) and HCl (1 mg/ml) under shaking at 130 rpm 146 at 25°C in the dark. Subcultures were inoculated with 4 mL of 5 day-old stock 147 suspensions (36). 148 Isolation and detection of crude protein 149 B. laterosporus strain A60 was cultured in 3000 ml LB medium with shaking at 180 rpm 150 for 48 h at 37°C, and the supernatant was collected after centrifugation (4700×g, 15 min, 151 4°C). Solid ammonium sulfate was added to the supernatant to achieve 80% relative 152 saturation (w/v) at 4°C overnight. The precipitate was harvested by centrifugation 153 (12,000×g, 20 min, 4°C), redissolved in 200 ml buffer A (25 mM MES–NaOH, pH 6.2) 154 and dialyzed against buffer A for 48 h. Before filtering the crude protein with a 0.22 μm 155 filter (Millipore, SuZhou, China), the insoluble debris was removed from the dialysate by 156 centrifugation (12,000 × g, 10 min, 4°C). A portion of the crude protein (50 μl) was tested 157 for elicitor activity (HR-inducing activity). 158 Purification of protein 159 Further purification was performed using the ÄKTA Explorer protein purification System 160 on O cber 7, 2017 by gest ht://aem .sm .rg/ D ow nladed fom (Amersham Biosciences). The crude protein was loaded onto an ion exchange 161 chromatography HiTrapTM SP FF column (GE Healthcare, Uppsala, Sweden) that was 162 pre-equilibrated with elution buffer (25 mM MES–NaOH, pH 6.2). The bound proteins 163 were eluted with a linear gradient of increasing NaCl in elution buffer at a flow rate of 2 164 ml/min. All fractions were collected and injected to a desalting column (GE Healthcare, 165 Uppsala, Sweden) for elicitor activity analysis. The purified protein was monitored for 166 elicitor activity. 167 The pooled active fraction after desalting was purified through High-Performance Liquid 168 Chromatography (HPLC) on a C18 reverse-phase column injected onto a Zorbax Eclipse 169 XDB-C18 reverse-phase column (150 × 4.6 mm, 5 μm, Agilent) equilibrated with 5% 170 ACN (acetonitrile)/2 mM NH4FA/0.1%FA/water. The pooled active fraction was eluted 171 with chromatography-grade ACN using a linear gradient of increasing from 20% (v/v) to 172 100% (v/v) over 30 min at a flow rate of 0.2 ml/min. All of the peaks were automatically 173 collected by the Fraction Collectors (Agilent). Each peak was freeze-dried, redissolved in 174 ultrapure water (Milli-Q, US), and tested for elicitor activity. The fraction with elicitor 175 activity was chromatographed again to ensure its purity, and the molecular weight was 176 determined via Tricine-SDS-PAGE. 177 Mass spectrometry analysis and gene identification 178 The protein sample was isolated on a Tricine-SDS-PAGE gel and digested overnight 179 using MS-grade Trypsin Gold (Promega, Madison, WI, USA). The digested peptides 180 on O cber 7, 2017 by gest ht://aem .sm .rg/ D ow nladed fom were reacted with succinimidyl-2-morpholine acetate (SMA) in order to analysis by 181 MS/MS. The purified peptides were sprayed into quadrupole time-of-flight (Q-TOF) 182 mass spectrometer (MicrO TOF-QII TM , Bruker Daltonics K.K., Tokyo, Japan) with an 183 electrospray ionization (ESI) ion source. The MS/MS data were automatically analyzed 184 by the MASCOT search engine (Matrix Science, London; http://www. 185 matrixscience.com), using the following parameters: Database, NCBInr; taxonomy, 186 B-laterosporus; enzyme, trypsin; Type of search, MS/MS Ion Search. The peptide and 187 fragment mass tolerance were set at 0.1 Da. Proteins with probability based MOWSE 188 scores exceeding the threshold (p<0.05) were definitely identified. 189 The genomic DNA was extracted from B. laterosporus strain A60 using an E.Z.N.A. TM 190 bacterial DNA Kit (Omega Bio-tek, Norcross, GA, USA). A pair of gene-specific primers 191 was designed to amplify the PeBL1 gene (GenBank accession no. KM668059) sequence 192 deduced from the mass spectrometry analysis and the Mascot database search. The primer 193 sequences were designed as follows: forward primer, 5’194 ATGAAAAAAGCTGTCTCAAC-3’ and reverse primer, 5’195 TTAGTAGGGAACAGTTATATT-3’. The PCR product was cloned into the pMD 18-T 196 vector (TaKaRa, Dalian, China) and verified by DNA sequencing (Beijing Genomics 197 Institution, Beijing, China). 198 Expression in E.coli and purification of recombinant protein 199 The PeBL1 gene, without its predicted signal peptide, was inserted into the 200 on O cber 7, 2017 by gest ht://aem .sm .rg/ D ow nladed fom pET30-TEV/LIC vector (Novagen, Darmstadt, Germany). Then, the recombinant plasmid 201 was transformed into the E. coli BL21 DE3 (TransGen Biotech, Beijing, China). The 202 primers, including a fragment of the pET30-TEV/LIC vector and the 5’ and 3’ ends of the 203 PeBL1 gene, were designed as follows: forward primer, 204 5’-TACTTCCAATCCAATGCCACACCAGCCAAACACTC-3’, and reverse primer 5’ 205 -TTATCCACTTCCAATGCTATTAGTAGGGAACAGTTATATTC-3’. The DNA was 206 isolated by electrophoresis and observed by staining with Gold View (SBS Genetech, 207 Beijing, China) and using Trans 2K DNA marker (TransGen Biotech, Beijing, China). 208 The PCR product was cloned into the vector using ligation-independent cloning (37), and 209 verified by DNA sequencing. 210 To express the recombinant PeBL1 protein, the bacteria were first grown at 37°C for 4 h, 211 and the recombinant protein was subsequently induced by the addition of 0.2 mM 212 isopropyl b-D-1-thiogalactopyranoside (Sigma, St. Louis, MO, USA) to the media at 213 16°C for 14-16 h. The acquired cells were disrupted two times with an ultrasonic 214 disruptor for pooling the supernatant. The mainly purification procedure of recombinant 215 PeBL1 as follows: affinity chromatography with a His-Trap HP column (GE Healthcare, 216 Waukesha, WI, USA), and a HiTrap desalting column (GE Healthcare, Waukesha, WI, 217 USA). The purified protein was tested for elicitor activity and detected by 218 Tricine-SDS-PAGE. Protein markers (Thermo Scientific, USA) were used to evaluate the 219 apparent molecular mass of the purified recombinant proteins. 220 on O cber 7, 2017 by gest ht://aem .sm .rg/ D ow nladed fom Characteristics of the PeBL1 protein 221 The HR-inducing activity of PeBL1 was evaluated in N. benthamiana leaves. The N. 222 benthamiana leaves was injected with samples (50 μl) or a control prepared by using a 223 syringe to cover areas of 1-2 cm 2 . The HR symptom was examined after 24 hours 224 according to the previously described method (38). The amplification of the tobacco HR 225 marker gene, HSR203, was performed with reverse transcription-polymerase chain 226 reaction (RT-PCR) with the following primers: HSR203 (forward, 227 5′-TGTACTACACTGTCTACACGC-3′; reverse, 228 5′-GATAAAAGCTATGTCCCACTCC-3′) and EF1a (forward, 5′229 AGACCACCAAGTACTACTGCAC-3′; reverse, 5′230 CCACCAATCTTGTACACATCC-3′) as a positive control. 231 In order to ascertain the influence of pH on elicitor activity, the pH of the elicitor was 232 adjusted to 4, 6, 8 or 11 with NaOH or HCl, incubated overnight, dialyzed, and then used 233 in the elicitor bioassay. 234 In order to determine elicitor heat stability, four aliquots of purified protein were 235 incubated at 4, 25, 50 and 75°C for 15 min, and subsequently the elicitor activity of the 236 treated proteins was tested. 237 Detection of hydrogen peroxide production and alkalinization of the extracellular 238 medium 239 The histological localization of hydrogen peroxide production in N. benthamiana leaves 240 on O cber 7, 2017 by gest ht://aem .sm .rg/ D ow nladed fom was determined as previously described (39). Briefly, PeBL1 (5 μM) or Tris-HCl 241 (negative control) was injected into 8-week-old leaves. Subsequently, the leaves were 242 isolated after 24 h of treatment and soaked in 3,3’-diaminobenzidine (DAB)-HCl (1 243 mg/mL, pH 3.8) solution. After incubation for 8 h in the dark, the treated leaves were 244 placed in 95% ethanol at 65°C to remove chlorophyll and observed under an Olympus 245 Stereomicroscope SZX9 (Olympus America, Inc., Melville, NY, USA). ROS production 246 in tobacco cell suspensions was quantified by chemiluminescence using luminol (40). In 247 brief, 250 μl cell were incubated with 300 μl of buffer (pH 5.75) containing 10 mM 248 HEPES, 175 mM mannitol, 0.5 mM CaCl2, 0.5 mM K2SO4 for 1 h at 26°C. Then PeBL1 249 (10 μM) and luminol (0.3 mM) were mixed into buffer and rotated in a shaker. The 250 chemiluminescence was expressed as nanomoles of H2O2 per gram fresh weight of 251 tobacco cells using a standard calibration curve and monitored by the GloMax-96 252 Luminometer (Promega, Madison, WI, USA). 253 The alkalinization of extracellular medium was performed in the tobacco cell suspensions 254 (41). The test was conducted simultaneously in 3×10 mL flasks (test, negative control and 255 positive control), each of which contained 1 g fresh weight (f.wt) of cells per 5 mL of cell 256 suspension. Tobacco cells were pre-equilibrated with an orbital shaker for approximately 257 45 min at 26°C until a steady pH value (5.0-5.2) was achieved and then treated with 258 PeBL1 (10 μM). The pH was observed for 90 min after PeBL1 addition. Flg22 (1 μM) 259 and buffer were added as a positive control and a negative control, respectively. The 260 on O cber 7, 2017 by gest ht://aem .sm .rg/ D ow nladed fom changes in the pH of the suspension mediums were monitored using a pH meter 261 (Sartorius Stedim, Germany). 262 Detection of phenolic compound accumulation in tobacco cell culture 263 For the measurement of phenolic compound accumulation in tobacco cells, 300 μl of 264 tobacco cell suspension was examined after incubation with 50 μl (10 μM) PeBL1 in the 265 dark at 26°C for 108 h under epifluorescence, Zeiss Axiovert 100 M inverted microscope 266 equipped with a confocal laser scanner (Zeiss LSM 510, Oberkochen, Germany). 267 Tris–HCl buffer (pH 7.3) was used as a negative control. 268 Bioassay for PeBL1-induced disease resistance in Nicotiana benthamiana 269 We used the TMV-GFP, a recombinant virus in which jellyfish green fluorescent protein 270 (GFP) gene was extended into the coat protein (CP) ORF of native TMV. The GFP was 271 visualized by using a 100-W long-wave UV lamp (Black Ray model B 100AP; UVP, 272 Upland, USA). The recombination did not influence the infection and movements of virus 273 in N. benthamiana (42, 43). Three leaves each from six plants were injected at one spot 274 per leaf with 10 μM PeBL1 or Tris–HCl buffer as a negative control. Three days later, the 275 upper three non-treated (systemic) leaves were inoculated with TMV-GFP. The 276 concentration of TMV-GFP solution was 0.5 g diseased leaves in 10 ml ultrapure water 277 (Milli-Q, US). The signs and diameter of TMV lesions in each leaf were analyzed at the 278 two, four, and six day post-inoculation (dpi) as previously described (44, 45). For the 279 diameter of the lesions, 10 randomly lesions were measured for each plant. Three 280 on O cber 7, 2017 by gest ht://aem .sm .rg/ D ow nladed fom replications were performed. The inhibition of TMV lesion was calculated using the 281 formula below. 282 Inhibition (%) = {(number(size)of lesions on control leaves-number(size)of lesions on 283 elictor-treated leaves))/ number(size)of lesions on control leaves}×100% 284 To assay whether PeBL1 can induce systemic resistance and diminish disease signs in N. 285 benthamiana, we next included a phytopathogenic bacteria (P. syringae pv. tabaci) in our 286 experiments. P. syringae pv. tabaci were cultivated at 28°C in King’s B media (46) for 24 287 h and harvested with centrifugation, then washed three times and resuspended in sterile 288 distilled water at an OD600 nm= 0.6 (1×10 7 CFU/ml). The method that N. benthamiana 289 plants were treated with PeBL1 and buffer was just as described in the assay of TMV 290 resistance. After three days, upper three non-treated leaves were inoculated with P. 291 syringae pv. tabaci by soaked for 45 s. Signs were assessed 4 days after challenge with P. 292 syringae pv. tabaci. Leaves were detached and sterilized to remove epiphytic bacterial 293 populations. Three samples were collected from each leaf with a sterilized hole punch and 294 ground with a pestle in 100 μl sterile water. The sample suspensions were vortexed 295 completely and serially diluted to 10 -3 . The bacteria in the dilution were inoculated on a 296 King’s B Kan 25 plate and grown for 2 d. The number of the colonies on each plate was 297 counted. The area of each sample was approximately 0.1963 cm 2 and CFU/cm 2 was 298 calculated by multiplying by the dilution factor. 299 Analysis of the expression of defense-related genes induced by PeBL1 using 300 on O cber 7, 2017 by gest ht://aem .sm .rg/ D ow nladed fom real-time quantitative PCR (RT-qPCR) 301 To study the mechanisms of the defense responses induced by PeBL1 in N. benthamiana 302 plants, N. benthamiana plants that were infiltrated with PeBL1 or buffer on three leaves 303 were assayed for the induction of several defense-related genes. A small fragment was 304 collected from the upper leaves at the indicated times and rapidly frozen in liquid 305 nitrogen. The fragments were placed in RNase-free tubes and frozen at -80 °C until use. 306 Control plants were infiltrated with buffer. Total RNA was extracted with the EasyPure TM 307 Plant RNA Kit (TransGen Biotech, Beijing, China). The cDNA was generated using the 308 TransScript TM All-in-one SuperMix for qPCR Kit (TransGen Biotech, Beijing, China), 309 and the concentrations of the cDNAs were adjusted to be equal. RT-qPCR was performed 310 to determine the relative expression levels of several defense-related genes and conducted 311 using the TransStart Green qPCR SuperMix UDG (TransGen Biotech, Beijing, China). 312 The specific genes were designed from the coding sequences of each gene using Beacon 313 Designer 8.12 (Table 1). The PCR was processed on an IQ-5 Real-Time System (Bio-Rad, 314 USA) under the following program: 50°C for 2 min, then 94°C for 10 min. followed 43 315 cycles of 94°C for 5 s, and 60°C for 30 s. A melting curve was established from 65 to 316 95°C. Three technical replicates were amplified for each sample, including negative 317 controls. The EF-1α (elongation factor 1α) gene was used as a reference gene for 318 normalization. The quantification of the relative changes in gene transcript level was 319 performed using the comparative 2-ΔΔCt method (47). The mean deviation was 320 on O cber 7, 2017 by gest ht://aem .sm .rg/ D ow nladed fom calculated from the standard deviation (SD) in the ΔΔCt value using the formula 2 -(ΔΔCt ± 321