Antifreeze protein from Ammopiptanthus nanus functions in temperature-stress through domain A

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Evaluation of AnAFP domains in cold resistance of E. coli

Two pairs of specific primers (Table S1) with restriction sites of EcoRI/BamHI were designed by using CE Design V1.04 (http://www.downcc.com/soft/281907.html), and used to amplify open reading frame (ORF) of AnAFP and four mutants deleting domain A (AnAFPΔA), K (AnAFPΔK), S (AnAFPΔS) and N (AnAFPΔN) created in our previous study, respectively. The products were cloned into the EcoRI/BamHI site of prokaryotic expression vector pINIII to generate pINIII-AnAFP, pINIII-AnAFPΔA, pINIII-AnAFPΔK, pINIII-AnAFPΔS and pINIII-AnAFPΔN, and confirmed by sequencing, respectively.

The re-constructed plasmids were transformed into competent cells of E. coli cold-sensitive strain BX04. After confirming by screening with 50 mg/L ampicillin, PCR amplification and sequencing, the positive colonies were transferred into LB liquid medium and incubated at 37 °C until OD600 = 0.5–0.6. According to the methods of Yang et al.34 and Deng et al.21 with minor modification, the cultured cells were treated at 17 °C for 9 days with three replicates. All samples were diluted by 100 to 107 times, respectively. The 5 μL and 100 μL of them were plated onto LB plates with 50 mg/L ampicillin, incubated at 37 °C for 12 h and photographed, respectively. Before treatment, 5 μL of them were plated onto LB plates with 50 mg/L ampicillin and incubated at 37 °C for 12 h for control. The colonies number with spraying 100 μL cells were counted and used to calculate average survival rates under cold stress.

Evaluation of AnAFP domains in heat resistance of E. coli

Two pairs of specific primers (Table S2) with restriction sites of NdeI/HindIII were designed, and used to amplify ORF of AnAFP, AnAFPΔA, AnAFPΔK, AnAFPΔS and AnAFPΔN using the above plasmids as template, respectively. The products were cloned into Nde I/Hind III site of prokaryotic expression vector pET28a to generate pET28a-AnAFP, pET28a-AnAFPΔA, pET28a-AnAFPΔK, pET28a-AnAFPΔS and pET28a-AnAFPΔN, and confirmed by sequencing, respectively.

The recombined plasmids were transferred into competent cells of E. coli BL21 (DE3). After confirming by screening with 50 mg/L kanamycin, and PCR amplification and sequencing, the positive colonies were transferred into LB liquid medium and incubated at 37 °C until OD600 = 0.5–0.6. The ectopic expression of AnAFP, AnAFPΔA, AnAFPΔK, AnAFPΔS and AnAFPΔN was induced by 0.5 mmol/L IPTG, detected by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). As described by Li et al.35 with minor modification, the induced cells were splinted into six centrifuge tubes, three of them were treated at 50 °C for 30 min, while others were incubated at 37 °C for 30 min, respectively. All samples were diluted by 100 to 104 times, respectively. The 5 μL and 100 μL of them were plated onto LB plates with 50 mg/L kanamycin, incubated at 37 °C for 12 h, and photographed. The colonies number with spraying 100 μL cells were counted and used to calculate average survival rate under heat stress.

Plasmids reconstruction and Arabidopsis transformation

Two pairs of specific primers (Table S3) with restriction sites of BspI/PstI were designed, and used to amplify ORF of AnAFP, AnAFPΔA, AnAFPΔK, AnAFPΔS and AnAFPΔN, respectively. The products were cloned into Bsp I/Pst I site of plants expression vector pCAMBIA2300-35SeGFP to generate 35SAnAFPeGFP, 35SAnAFPΔAeGFP, 35SAnAFPΔKeGFP, 35SAnAFPΔSeGFP and 35SAnAFPΔNeGFP, and confirmed by sequencing, respectively. These plasmids were transferred into competent cells of Agrobacterium tumefaciens GV3101 by freeze–thaw method.

After confirming by screening with 50 mg/L rifampicin and 50 mg/L kanamycin on YEB plates, PCR amplification and sequencing, the positive colonies were transferred into liquid medium YEB and incubated at 28 °C until OD600 = 1.0–1.5. The cells were collected by centrifugation at 4 °C and 4000 r/min for 10 min, resuspended and adjusted to OD600 = 1.0 with 5% (wt/vol) fresh sucrose solution, added surfactant Silwet L-77 to a concentration of 0.02% (vol/vol), and used to transform Arabidopsis mutant of the KnS type dehydrin gene AtHIRD11 (AT1G54410) by floral-dip method.

Heat tolerance of transgenic Arabidopsis

As described by Sun et al.36, T1 seeds were surface-sterilized with 75% ethanol for 1 min and 10% NaClO for 10 min, and plated onto 1/2 MS plates with 50 mg/L kanamycin (Sigma, USA) for screening of transgenic plants, which were used to produce T2 generation. The T2 plants with 3:1 segregating-ratio to resistance/susceptibility of kanamycin were self-pollinated to generate T3. The homozygous lines without segregation were collected from T3, and were identified by PCR amplification using the primers (Table S4) for the specific fragments of AnAFP, AnAFPΔA, AnAFPΔK, AnAFPΔN and AnAFPΔS. The total RNA of every line was extracted by RNA extractor kit (Sangon, China), reacted with RNase-free DNase I, and used to reverse transcribed into cDNA using PrimeScript RT Reagent Kit (TaKaRa, Dalian). The ectopic expression of the transformed genes was identified by reverse transcription PCR (RT-PCR) using the above primers. The AtActin gene was amplified and used as reference. Five T3 lines were planted in pots, and grown in green house at 22 ℃ and 60–70% relative humidity under a 10 h light/14 h dark photoperiod. One-month-old seedlings were used for heat-shock treatment at 46 °C for 3 h, and recovered for 2 weeks at 22 ℃, and investigated for phenotype.

Subcellular localization

The 35SAnAFPeGFP, 35SAnAFPΔAeGFP, 35SAnAFPΔKeGFP, 35SAnAFPΔSeGFP and 35SAnAFPΔNeGFP plasmids were precipitated onto 50 mg of 0.6 µm gold particles by 2.5 mol/L CaCl2 and 0.1 mol/L spermidine, respectively, and used for transformation of intraepidermal cells of onion bulbs by microprojectile bombardment on DuPont PDS 1000/He (Bio-Rad, USA). The green fluorescence signal was observed using laser confocal microscope LSM 800 (Carl Zeiss, Germany).

Yeast two-hybrid

Two pairs of specific primers (Table S5) with restriction sites of NdeI/BamHI were designed, and used to amplify ORF of AnAFP, AnAFPΔA, AnAFPΔK, AnAFPΔS and AnAFPΔN, respectively. The products were cloned into NdeI/BamHI sites of yeast two hybrid (Y2H) bait vector pGBKT7 to generate pGBKT7-AnAFP, pGBKT7-AnAFPΔA, pGBKT7-AnAFPΔK, pGBKT7-AnAFPΔS and pGBKT7-AnAFPΔN, and confirmed by sequencing, respectively. Another pair of specific primers (Table S6) with restriction sites of NdeI/BamHI were designed and used to amplify ORF of AnICE1 from the cDNA of A. nanus seedlings. The products were cloned into Y2H trap vector pGADT7 to generate pGADT7-AnICE1 and confirmed by sequencing.

The yeast strain Y2H Gold was transformed with every plasmid using Yeastmaker Yeast Transformation System 2 (Clotech, Japan), and used to test of self-activation and toxicity of AnAFP, AnAFPΔA, AnAFPΔK, AnAFPΔN, AnAFPΔS, and AnICE1 proteins. Subsequently, the yeast strain Y2H Gold was co-transformed by each pair of bait vector pGBKT7-AnAFP, pGBKT7-AnAFPΔA, pGBKT7-AnAFPΔK, pGBKT7-AnAFPΔN, pGBKT7-AnAFPΔS, and trap vector pGADT7-AnICE1, respectively. The transformants were screened on the auxotroph SD/-Trp-Leu plates at 30 °C for 3–5 days. The mono-colonies were transferred onto the auxotroph SD/-Leu-Trp-His-Ade plates, incubated at 30 °C for 3–5 days, and strained with X-α-Gal.

Real-time quantitative PCR

The seeds of A. nanus were surface-sterilized with 75% ethanol for 10 min, and planted in soil and grown in green house at 25 °C 12 h light/ 20 °C 12 h dark and 60–70% relative humidity. At the six-leaf stage, the seedlings with same size were treated at high temperature of 45 °C for 0 (negative control), 3, 6, 12, and 24 h with three biological replicates, as described by Yu et al.37. The total RNA was extracted, and reverse transcribed into cDNA as above.

For pairs of specific primers (Table S7) were designed, and used to amplify a 150–250 bp fragment for AnAFP, AnAFPΔA, AnAFPΔK, AnAFPΔN and AnAFPΔS, as well as the internal reference gene AnGAPDH. The RT-qPCR was performed using SYBR Green Super Mix (Bio-Rad, USA) by two step real-time PCR cycles (95 °C 30 s; 95 °C 5 s, 50–65 °C 30 s, 39 cycles) in CFX96 Real-Time System (Bio-Rad, USA). The 2−ΔΔCT method was used to normalize the expression differentiation between reference gene and investigated genes38.

Statistical analysis

All experiments were conducted with three replicates. The data are presented as the mean values ±  standard deviation (SD). Statistical difference was analyzed using Student’s t tests.

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