Cloning and analysis of OsCYP2 promoter
Seeds of wild type (O. sativa L. cv. Aichi-ashahi, kindly provided by Institute of Biotechnology, Hangzhou Academy of Agricultural Sciences) were germinated with distill water in dark incubator at 30 °C and cultured at 32 °C/28 °C with the photoperiod of 16 h/8 h (light/dark). The total DNA of seeding was extracted from wild type according to modified CTAB method. An upstream 1156 bp fragment of the OsCYP2 was amplified by specific primers (Supplementary Table S1) and then used to substitute the promoter of GUS in pCAMBIA1301 to construct a pOsCYP2:GUS plasmid (Supplementary Fig. S1). The cis elements of the promoter were predicted by PLACE (Higo et al., 1999).
Histochemical GUS assay of OsCYP2
The pOsCYP2:GUS vector was transformed into wild type by Agrobacterium tumefaciens strain EHA105 according to previously reference described with minor modification (Hiei et al., 1994). GUS activity was conducted with various tissues (panicle, leaves, stem and root) of wild type and transformants via histochemical staining by 0.5 mg/mL X-gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronide), which contained 80 mM phosphate buffer, 0.5 mM K3Fe(CN)6, 0.5 mM K4Fe(CN)6, 0.1% Triton X-100, and one drop N,N-dimethyl formamide. All the samples were incubated at 37 °C for 24 hr, and rinsed 30 min with a series of 50%, 75%, 100% ethanol, respectively. Then, these samples were kept in 75% ethanol for analysis and photograph.
Construction of bait yeast strain and testing for AbA expression
Based on cis elements of the promoter, ABA responsive element (ABRE) target sequence (p62) was used as a bait and mutant sequence (p63) as a negative control simultaneously. Two antiparallel oligonucleotides sequence (Supplementary Table S1) of ABRE cis element with HindIII and XhoI overhanging sticky ends was synthesis, then anneal the oligonucleotides and ligation into the linearized pAbAi vector. Recombinant plasmids were linearized through BstBI and transformed into Y1H for generating bait reporter yeast strains. The minimal inhibitory concentration of AbA was conducted to bait reporter yeast strains.
Yeast one-hybrid assay
The pre-constructed cDNA library was transformed into Y1H bait strain (pBait-AbAi) using the Yeastmaker Transformation System 2 (Clontech, Cat. No. 630439) (Cui et al., 2016). The transformation reaction was spread on SD/-Leu plates with plus minimal inhibitory concentraion of AbA. The candidate clones were restreaked onto SD/-Leu/AbA at least three times to generate single colonies. For eliminate duplicate clones, we conducted yeast colony PCR by Matchmaker Insert Check PCR Mix 2 (Clontech, Cat. No. 630497). The prey vectors that showed single band in yeast colony PCR was isolated by Easy Yeast Plasmid Isolation Kit (Clontech, Cat. No. 630467) and sequenced using T7 primer. To verify positive interactions, each prey should be transformed into bait and mutant bait strain on selective media with side by side positive and negative controls.
Electrophoretic mobility shift assay
The transcription factor myc2-like was cloned into pET28a for electrophoretic mobility shift assays (EMSA). The recombinant vector was transformed in E. coli BL21 competent cells and fusion protein was purified by Magnehis Protein Purification Kit (Promega, Cat. No. V8500). Analyze the samples by 12% SDS-PAGE and western blot with His tag antibody conjugated horseradish peroxidase. The oligonucleotides containing three tandem copies of the elements were listed in Table S1 and labelled by Biotin 3’ End DNA Labelling Kit (Thermo, Cat. No. 89818). Electrophoretic mobility shift assays were conducted using Light Shift Chemiluminescent EMSA Kit (Thermo, Cat. No. 20148) according to the manufacturer’s instructions.
Transcript level of myc2-like under salt and ABA induction
Seedlings of wild type were cultivated as previously conditions. Samples were treated by 150 mM NaCl and 50 μM ABA. Transcript level of myc2-like was conducted by qRT-PCR after treated 2 hr, 12 hr and 24 hr. Three replications and statistical analysis of experiments were calculated according to previous described. The primers of myc2-like was listed in Supplementary Table S1. Each sample was performed three replicates and calculated according to the method of Livak and Schmittgen 2001.
Construction of myc2-like overexpression transformants in wild type and cyp2-RNAi line
The recombinant vector of p1300-Ubiquitin-myc2-like was constructed, then transformed into wild type and cyp2-RNAi line L3-61 (Cui et al., 2017) through A. tumefaciens strain EHA105 to create overexpression transformants. Plant transformation was performed according to previously reference described with minor modification (Higo et al., 1999). Independent transformants of myc2-like overexpression in wild type and cyp2-RNAi line were identified by quantitative RT-PCR.
Physiological and biochemical assays of transformants
To evaluate the salt response of transformants, seedlings of wild type, myc2-like overexpression in wild type, myc2-like overexpression in cyp2-RNAi and cyp2-RNAi were treated by 150 mM NaCl for 24 hr. Various physiology and biochemistry index were measured according to Cui et al., 2016, including superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), malondialdehyde (MDA) and proline. Sodium and potassium ions contents were analyzed through atomic absorption spectrophotometry (Munns et al., 2010).
Statistical analysis of the results presented here are the means of at least three replicates. Treatment means were compared by the analysis of variance (ANOVA) and using the least significant difference (LSD) test at the P≤0.05 level of significance.