Gene 549 (2014) 77–84
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Heterologous expression of betaine aldehyde dehydrogenase gene from Ammopiptanthus nanus confers high salt and heat tolerance to Escherichia coli Hao-Qiang Yu a,1, Ying-Ge Wang a,1, Tai-Ming Yong a, Yue-Hui She b, Feng-Ling Fu a,⁎, Wan-Chen Li a,⁎ a b
Maize Research Institute, Sichuan Agricultural University, Chengdu, Sichuan 611130, PR China Agronomy Faculty, Sichuan Agricultural University, Chengdu, Sichuan 611130, PR China
a r t i c l e
i n f o
Article history: Received 18 May 2014 Received in revised form 17 June 2014 Accepted 17 July 2014 Available online 18 July 2014 Keywords: Ammopiptanthus nanus Betaine aldehyde dehydrogenase Heat Heterologous expression Salt Tolerance
a b s t r a c t Betaine aldehyde dehydrogenase (BADH) catalyzes the synthesis of glycine betaine, a regulator of osmosis, and therefore BADH is considered to play a significant role in response of plants to abiotic stresses. Here, based on the conserved residues of the deduced amino acid sequences of the homologous BADH genes, we cloned the AnBADH gene from the xerophytic leguminous plant Ammopiptanthus nanus by using reverse transcription PCR and rapid amplification of cDNA ends. The full-length cDNA is 1868 bp long without intron, and contains an open reading frame of 1512 bp, and 3′- and 5′-untranslated regions of 294 and 62 bp. It encodes a 54.71 kDa protein of 503 amino acids. The deduced amino acid sequence shares high homology, conserved amino acid residues and sequence motifs crucial for the function with the BADHs in other leguminous species. The sequence of the open reading frame was used to construct a prokaryotic expression vector pET32a-AnBADH, and transform Escherichia coli. The transformants expressed the heterologous AnBADH gene under the induction of isopropyl β-D-thiogalactopyranoside, and demonstrated significant enhancement of salt and heat tolerance under the stress conditions of 700 mmol L−1 NaCl and 55 °C high temperature. This result suggests that the AnBADH gene might play a crucial role in adaption of A. nanus to the abiotic stresses, and have the potential to be applied to transgenic operations of commercially important crops for improvement of abiotic tolerance. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Drought, salinity, extreme temperatures and other abiotic stresses significantly restrict productivity and quality of crops (Hu and Xiong, 2014; Roy et al., 2014; Tambo and Abdoulaye, 2012), which adapt themselves to these abiotic stresses through a series of mechanisms (Dong et al., 2014; Neumann, 2008; Zheng et al., 2010). Compatible solutes, such as proline, glycine betaine and soluble sugars play a crucial role in the process counteracting stress via balancing osmotic potential (Chen and Murata, 2011; Dong et al., 2014; Silvente et al., 2012). Glycine betaine is an important osmoprotectant that can stabilize the structure and function of biomembrane system, enzymes, photosystem II complexes, ribulose-1,5-bisphosphate carboxylase/oxygenase, and many other functional proteins (Bao et al., 2011; Carillo et al., 2008, 2011; Chen and Murata, 2008; Gill et al., 2014; Prasad and Saradhi, 2004). In Abbreviations: RACE, rapid amplification of cDNA ends; BADH, betaine aldehyde dehydrogenase; ORF, open reading frame; CTAB, hexadecyltrimethylammonium bromide; UTR, untranslated region; IPTG, isopropyl β-D-thiogalactopyranoside; LB, Luria–Bertani; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. ⁎ Corresponding authors. E-mail addresses: ffl@sicau.edu.cn (F.-L. Fu), [email protected] (W.-C. Li). 1 Contributed equally.
http://dx.doi.org/10.1016/j.gene.2014.07.049 0378-1119/© 2014 Elsevier B.V. All rights reserved.
plants, the biosynthesis of glycine betaine consists of two steps: choline oxidation to betaine aldehyde catalyzed by choline monooxygenase, and betaine aldehyde to glycine betaine via deoxygenation catalyzed by betaine aldehyde dehydrogenase (BADH) (Rasheed et al., 2011). The heterologous overexpression of betaine aldehyde dehydrogenase genes increased betaine aldehyde accumulation, and improved tolerance of the transgenic plants to drought, salinity, extreme temperatures and other abiotic stresses (Fan et al., 2012; Karabudak et al., 2014; Li et al., 2011, 2014; Wu et al., 2008; Zhang et al., 2012; Zhou et al., 2008). However, the different improved phenotypes were obtained from these transgenic events. Except for the differentiation of gene expression, the activities of the BADHs encoded by the transformed exogenous genes themselves were probably one of the major reasons. Therefore, cloning and function evaluation of new betaine aldehyde dehydrogenase genes, particularly from abiotic stress-tolerant plant species, become helpful for transgenic operation of commercially important crops for improvement of abiotic tolerance. Ammopiptanthus nanus, one of the two relict species of the Ammopiptanthus genus (Leguminosae), is the unique evergreen broadleaf bush in the plateau desert from the west edge of the Tarim Basin to the border between China and Kyrgyzstan (Cheng, 1959). It is endemic to the harsh environments of arid climate (annual precipitation
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b 200 mm), extreme temperatures (from − 30 °C to 40 °C), dryness (dryness usually more than 4), poor soil quality and high salinity (the habitats are usually stony and/or sandy slopes) (Pan et al., 1992; Wang, 2005; Wang et al., 2007; Yan et al., 2000). According to the criteria that 80%–100% of leaves are severely affected (Miller, 1963), the critical high temperature of A. nanus is 65 °C, significantly higher than other sandy plants (Liu and Qiu, 1982; Liu et al., 1995). A germination test showed that the critical concentration of NaCl that permits the germination of A. nanus seeds was 1.38% (Wang and Yin, 1991). Another study of our research team has demonstrated that the antifreeze protein gene of A. nanus has stronger function than those cloned from other plants (Deng et al., 2014). Therefore, it is promising to clone and evaluate other stress-related genes from this tenacious species for understanding the molecular mechanism of tolerance to abiotic stress and probable application in transgenic operation. This paper reports the cloning of the AnBADH gene from A. nanus and the function evaluation by heterologous expression in Escherichia coli.
2. Materials and methods 2.1. Plant material and NaCl treatment The seeds of A. nanus were soaked in 60 °C water for 5 min, and sown in sterilized nutrient soil. The seedlings were grown at 26/30 °C under the light/dark cycle of 12/12 h for four weeks, and then subjected to salt stress by adding 500 mmol L − 1 NaCl, in order to increase the expression of the AnBADH gene. Five hours later, the leaves were harvested, snap frozen in liquid nitrogen and stored at − 80 °C.
2.2. Cloning of full-length cDNA Total RNA was extracted from the stored leaf sample by using RNAiso plus kit (TaKaRa, Japan), and treated with RNase-free DNase to remove probable genomic DNA. The first strand of the cDNA was reverse transcribed by using the total RNA sample as the template, and PrimeScript™ II 1st Strand cDNA Synthesis kit (TaKaRa, Japan). A pair of primers (5′-CTGTGAAT GGCGACACGGAAG-3′/5′-GATTGTTCCACGCTCGCTCTTAG-3′) was designed according to the partial cDNA sequence (GenBank accession number: DQ288723) of the BADH gene in Ammopiptanthus mongolicus, the other species of the Ammopiptanthus genus, and used to amplify the core sequence of the AnBADH gene with the sample of the first strand of the cDNA, and high fidelity DNA polymerase (TaKaRa, Japan). The first strand of the cDNA for 3′-RACE (rapid amplification of cDNA ends) was reverse transcribed by using the total RNA as the template, and the 3′-RACE adaptor with 3′-Full RACE Core Set Ver.2.0 (TaKaRa, Japan). The product was used for the first amplification of the nested PCR with the specific outer primer (5′-ACTGGAAGCTCTGCAACTGGGA CCAAGA-3′) designed according to the core sequence, and the 3′RACE outer primer (5′-TACCGTCGTTCCACTAGTGATTT-3′). The product was used for the second amplification of the nested PCR with the specific inner primer (5′-TCAAGCCTGTTTCACTAGAGCTCGGTGG-3′) designed according to the core sequence, and the 3′-RACE inner primer (5′-CGCG GATCCTCCACTAGTGATTTCACTATAGG-3′). The 5′-RACE was conducted using First Choice® RLM-RACE kit (Ambion, USA). The total RNA sample was dephosphorylated with calf intestinal alkaline phosphatase, decapped with tobacco acid pyrophosphatase, ligated with the 5′ RACE adaptor, and used for the reverse transcription of the first strand of the cDNA with the random
Fig. 1. Prokaryotic expression vector pET32-AnBADH. The AnBADH gene is under the control of the promoter and terminator of phage T7. The sequence immediately after the promoter encodes a leader peptide including a sulfoxide reductase protein tag (Trx-tag), two histidine selective tags (His-tag) and an S-tag for western blotting. The ampicillin resistance gene Ap is used as the selective marker.
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Fig. 2. Full-length cDNA sequence of AnBADH gene in A. nanus. The frame and asterisk indicate the start codon and the stop codon, respectively.
decamer primer. The product was used for the first amplification of the nested PCR with the specific outer primer (5′-GCACCAGCTTCA GGACCTAATCCAGTGA-3′) designed according to the core sequence, and the 5′-RACE outer primer (5′-GCTGATGGCGATGAATGAACAC TG-3′). The product was used for the second amplification of the nested PCR with the specific inner primer (5′-CCAGCTCCAAACAG GTCACAGATGCCAA-3′) designed according to the core sequence, and the 5′-RACE inner primer (5′-CGCGGATCCGAACACTGCGTTTGC TGGCTTTGATG-3′).
The amplified products of the core sequence, 3′-RACE, and the 5′-RACE were subcloned into the pMD19-T vector (TaKaRa, Japan), sequenced at Invitrogen (USA) and Sangon (China), repeatedly, and assembled into the full-length cDNA sequence. 2.3. Amplification of genomic DNA sequence Total DNA was extracted from stored leaf sample by the method of CTAB (Doyle and Doyle, 1987), and used to amplify the genomic DNA
Fig. 3. Phylogenetic tree among the putative and the BADH proteins in other plants.
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Fig. 4. Five crucial domains of AnBADH and four other BADH proteins. The asterisk, double and single-spot indicate perfect (100%), high (75%), and low (25%) conservation of the amino acids, respectively. The gray backgrounds, black triangle and solid circles indicate NAD (P)+ binding sites, catalytic residues, and the transit signal, respectively.
sequence of the AnBADH gene with a pair of specific primers (5′-ATGG CAA CCCCAATACCGAAT-3′ /5′-CCTATCACAGCTTTGAAGGAGACTG-3′) designed according to the full-length cDNA sequence, and Prime STAR HS DNA Polymerase (TaKaRa, Japan). The product was added with an adenosine triphosphoric acid to each of the 3′-ends by using Taq DNA Polymerase (Tiangen, China), subcloned into the pMD19-T vector, sequenced at Invitrogen (USA) and Sangon (China), repeatedly, and used for alignment with the full-length cDNA sequence.
ORF sequence by using Prime STAR HS DNA Polymerase (TaKaRa, Japan). The product was digested by BamH I/Hind III, and inserted into a prokaryotic expression vector pET32a (Fig. 1). The E. coli strain Rosetta (DE3) was transformed by the loaded expression vector pET32aAnBADH and empty prokaryotic pET32a (negative control), and screened on a Luria–Bertani (LB) agar plate containing 50 mg L− 1 ampicillin at 37 °C overnight. 2.6. Induced expression and stress treatment
2.4. Bioinformatics analysis The open reading frame (ORF) of the AnBADH gene was identified from the full-length cDNA sequence by online software ORF finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). The putative amino acid sequence was deduced online (http://web.expasy.org/translate/) from the full-length cDNA sequence of the AnBADH gene, and used for multiple alignment with BADHs of relative and model species by using ClustalX2.1 software (http://mac.softpedia.com/get/Math-Scientific/ ClustalX.shtml). The physical and chemical properties, secondary structure, transmembrane regions and orientation, subcellular location and signal peptide were predicted by using Expasy (http://www.expasy. org/), GOR IV (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page= npsa_gor4.html), TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/ services/TMHMM-2.0/), WoLF PSORT (http://wolfpsort.seq.cbrc.jp/), and SignalP 4.1 (http://www.cbs.dtu.dk/services/SignalP/), respectively. The phylogenetic tree was constructed by using software MEGA5.1 (http://mega.software.informer.com/5.1b/). The conserved domains were analyzed by using Conserved Domains Search Tool (http://www. ncbi.nlm.nih.gov/cdd/). 2.5. Transformation of E. coli A pair of specific primers (5′-GTGGATCCATGGCAACCCCAATACCG AAT-3′/5′-CATCAAGCTTCCTATCACAGCTTTGAAGGAGACTG-3′) was designed with the introduction of the recognition sites of BamH I and Hind III (the underlined bases in the bracket), and used to amplify the
After confirmation by bacterial PCR and double digestion of the extracted plasmids with BamH I/Hind III, the positive single colonies were cultured in LB liquid medium containing 50 mg L−1 ampicillin at 37 °C. After 5–6 h until OD600 ≈ 0.6, the cultures were added with isopropyl β-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mmol L−1, and incubated at 37 °C for 4 h. Then, each of the cultures was adjusted to OD600 ≈ 0.6 with the ampicillin LB liquid medium, and divided into five portions. From each of the first portions, the E. coli cells were harvested, resuspended in 1× PBS buffer and 2 × SDS sample buffer, lysed in a boiling water bath for 5 min, centrifuged at 13,000 r/min for 10 min, and used for the detection of the heterologous expression of the AnBADH gene by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Referring to He et al. (2012) and Udawat et al. (2014), each of the second portions was diluted by ten-fold serial to 1:105 with the ampicillin LB liquid medium. Five microliters of each of the dilutions was spotted onto the ampicillin LB agar plates containing NaCl of 170 (control), 300, 500, 700 and 1000 mmol L−1. After incubation at 37 °C for 16 h, the growth status of the colonies was observed, and used to determine the suitable NaCl concentration for high salt treatment. One milliliter was taken from each of the third portions, and added into 20 mL of the ampicillin LB liquid medium containing NaCl of the suitable concentration for high salt treatment. OD600 value was measured at every hour during incubation at 37 °C, and used for the analysis of the growth dynamics.
Fig. 5. Conserved domains of the putative protein. A NAD (P)+-dependent ALDH-SF and NAD (P)+ binding sites are included.
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Each of the fifth portions was subjected to heat treatment of 55 °C for the suitable duration, and diluted to 1:108 with the ampicillin LB liquid medium. One hundred microliters was taken from the dilutions, and spread onto the ampicillin LB agar plates with three replicates. After incubation at 37 °C for 16 h, colonies were counted, and used to calculate the average survivals of the three replicates. 3. Results 3.1. Full-length cDNA sequence The amplified core sequence was 585 bp in length, and shared high similarity to the BADH genes in other leguminous plants. 1046 bp and 654 bp fragments were amplified with the inner primers in the second amplification of the 3′-RACE and 5′-RACE, respectively. The overlapping regions of these three sequences were perfectly matched, and assembled into a full-length cDNA of 1868 bp long, with a 5′-untranslated region (UTR) of 62 bp, an open reading frame (ORF) of 1512 bp, and a 3′UTR of 294 bp (Fig. 2). The alignment with the genomic DNA sequence showed that there was no intron in the genomic DNA sequence. Fig. 6. Heterologous expression of AnBADH gene in E. coli fractionated by SDS-PAGE. Lane M: molecular weight marker; lane 1: uninduced transformant by empty vector pET32a; lane 2: IPTG-induced transformant by empty vector pET32a; lane 3: uninduced transformant by loaded vector pET32a-AnBADH; lane 4: IPTG-induced transformant by loaded vector pET32a-AnBADH, the biggest band is the fusion protein of the leader peptide and the AnBADH.
Referring to He et al. (2012) and Wu et al. (2012), each of the fourth portions was subjected to a heat treatment of 55 °C. At the 0th (control), 5th, 10th, 15th, 30th, 45th and 60th min, the cultures were sampled, and diluted by ten-fold serial to 1:105 with the ampicillin LB liquid medium. Five microliters of each of the dilutions was spotted onto the ampicillin LB agar plates. The growth status of the colonies was observed after incubation at 37 °C for 16 h, and used to determine the suitable duration of heat treatment.
3.2. Properties of putative protein The putative protein of the 1512 bp ORF contains 503 amino acids, with 54.71 kDa, isoelectric point pH 5.39, instability index (II) 28.36 (b40), and grand average of hydropathicity (GRAVY) − 0.015 (b0). The percentages of α-helices, ß-sheets and random coils in the secondary structure were predicted to be 41.4%, 7.6% and 34.19%, respectively. Its subcellular localization was predicted to be chloroplast. These properties suggest that the putative protein is a stable hydrophilic protein, neither a transmembrane protein nor a secreted protein. The multiple alignment showed that the amino acid sequence of the putative protein shares the highest identity with BADHs of leguminous plants, such as Glycine max (GenBank accession no. ADN03184.1) and Phaseolus vulgaris (GenBank accession no. XP_007138125.1) (Fig. 3). Particularly, the transit signal Ser-Lys-Leu (SKL) at the C-terminal (Fig. 4), the conserved domains
Fig. 7. Growth status of the E. coli colonies transformed by pET32a-AnBADH and pET32a under different salt concentrations.
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Fig. 8. Growth status of the colonies transformed by pET32a-AnBADH and pET32a after 55 °C heat treatment for different times.
of the nicotinamide adenine dinucleotide phosphate-dependent aldehyde dehydrogenase superfamily [NAD(P)+-dependent ALDH-SF] and the nicotinamide adenine dinucleotide phosphate [NAD (P)+] binding sites are essential for the function of BADH (Figs. 4 and 5) (Diaz-Sanchez et al., 2012; Reumann, 2004). All these properties identify the putative protein as a betaine aldehyde dehydrogenase, and its encoding sequence as the AnBADH gene. Therefore, we registered this sequence at GenBank with accession number KJ841914.
3.3. Heterologous expression of AnBADH gene confers high salt and heat tolerance to E. coli The result of SDS-PAGE revealed an obvious additional band about 76 kDa present in the IPTG-induced transformed line by the loaded vector pET32a-AnBADH, but absent in the other three negative controls, indicating the heterologous expression of the AnBADH gene in the transformed E. coli line (Fig. 6). Figs. 7 and 8 show the gradual decrease of the colony number on the LB agar plates along with the increase of the salt concentration, duration of the heat treatment, as well as the dilution fold. However, obvious differences were present between the lines transformed by the loaded vector pET32a-AnBADH and by the empty vector pET32a. The suitable NaCl concentration for high salt treatment is 700 mmol L−1, and the suitable duration of the 55 °C heat treatment is 15 min. Fig. 9 shows the growth dynamics of the transformed lines by the loaded vector pET32a-AnBADH and the empty vector pET32a. In the control medium (170 mmol L−1 NaCl), the growth speed of these two lines was not significantly differential, and entered the exponential growth phase in a short time. In the high salt medium (700 mmol L−1 NaCl), however, the growth of the transformed line by the loaded vector pET32a-AnBADH increased significantly faster than the transformed line by the empty vector pET32a. The range of this differentiation increased with time. As shown in Fig. 10, the colony number of the transformed line by the loaded vector pET32a-AnBADH on the ampicillin LB agar plates was obviously more than that of the transformed line by the empty vector pET32a. The average survival rates of the three replicates were 45.3% for the transformed lines by the loaded vector pET32a-AnBADH, and 29.8% for that by the empty vector pET32a. This differentiation was proven to be significant by t-test (p b 0.01). 4. Discussion
Fig. 9. Growth curves of the transformed lines by pET32a-AnBADH and pET32a in the control (170 mmol L−1 NaCl) and the high salt (700 mmol L−1 NaCl) mediums. 1: IPTGinduced transformant by loaded vector pET32a-AnBADH in the control mediums; 2: IPTG-induced transformant by empty vector pET32a in the control mediums; 3: IPTGinduced transformant by vector pET32a-AnBADH in the high salt mediums; 4: IPTGinduced transformant by empty vector pET32a in the high salt mediums.
There was a fusion gene on the pET32a vector that encodes a 15.3 kDa fusion leader peptide including a sulfoxide reductase protein tag (Trx), two histidine selective tags (His) and an S tag for western blotting (Fig. 1). Its overexpression induced by IPTG might cause metabolic burden and inhibit the expression of the endogenous proteins in E. coli. This was demonstrated by the weaker background bands in lanes 2 and 4 loaded with the IPTG-induced lysates than those in lanes 1 and 3
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Fig. 10. E. coli colonies transformed by pET32a-AnBADH and pET32a after 55 °C heat treatment for 15 min. I, II, III indicate three replicates, respectively.
loaded with the uninduced lysates in Fig. 6, and by the similar results in the transformed E. coli lines by other heterologous genes (Aris et al., 1998; Deng et al., 2014; Kurland and Dong, 1996; Li et al., 2013; Ricci and Hernandez, 2000). Therefore, we did not conduct IPTG induction in the incubation for high salt and heat treatment. The additional band in lane 3 loaded with the uninduced lysate, which displayed the same migration rate as the biggest band in lane 4 loaded with the IPTG-induced lysate, demonstrated the heterologous expression of the AnBADH gene without IPTG induction (Fig. 6). The results of the present study indicated that the heterologous expression of the AnBADH gene from A. nanus confers high salt and heat tolerance to E. coli, which has been sufficiently evaluated to be an effective heterologous expression system for plant genes (He et al., 2012; Lan et al., 2005; Li et al., 2013; Wu et al., 2012), and particularly for genes from the Ammopiptanthus genus (Deng et al., 2014; Liu et al., 2010). The heterologous expression of the BADH genes from other plants has been reported to increase the accumulation of glycine betaine, and enhance the tolerance of E. coli to abiotic stresses (Yang et al., 2012; Yilmaz and Bülow, 2002). The heterologous expression of the BADH genes from other plants also resulted in improvement of the transgenic plants for abiotic stresses (Fan et al., 2012; Karabudak et al., 2014; Li et al., 2011, 2014; Wu et al., 2008; Zhang et al., 2012; Zhou et al., 2008). Therefore, the results of the present study suggest that the expression of the AnBADH gene and the accumulation of glycine betaine are two of the molecular mechanisms of how A. nanus copes with the harsh environments. These mechanisms imply the potential of the AnBADH gene to be applied to genetic modification of commercially important crops for improvement of abiotic tolerance.
5. Conclusion The full-length cDNA of the AnBADH gene from A. nanus is 1868 bp long without intron, and contains an open reading frame of 1512 bp, encoding a 54.71 kDa protein of 503 amino acids. Its heterologous expression confers high salt and heat tolerance to E. coli, suggesting that the AnBADH gene might play a crucial role in adaption of A. nanus to the abiotic stresses, and have the potential to be applied to transgenic operations of commercially important crops for improvement of abiotic tolerance.
Conflict of interest There is no conflict of interest in the submission of this manuscript.
Acknowledgments This work was supported by the National Key Science and Technology Special Project (2013ZX08003-005), and the National Natural Science Foundation of China (31071433). The authors thank the technical support from the Key Laboratory of Biology and Genetic Improvement of Maize in Southwest Region, and the anonymous reviewers for their critical reading and modification suggestion.
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Gene journal homepage: www.elsevier.com/locate/gene
Heterologous expression of betaine aldehyde dehydrogenase gene from Ammopiptanthus nanus confers high salt and heat tolerance to Escherichia coli Hao-Qiang Yu a,1, Ying-Ge Wang a,1, Tai-Ming Yong a, Yue-Hui She b, Feng-Ling Fu a,⁎, Wan-Chen Li a,⁎ a b
Maize Research Institute, Sichuan Agricultural University, Chengdu, Sichuan 611130, PR China Agronomy Faculty, Sichuan Agricultural University, Chengdu, Sichuan 611130, PR China
a r t i c l e
i n f o
Article history: Received 18 May 2014 Received in revised form 17 June 2014 Accepted 17 July 2014 Available online 18 July 2014 Keywords: Ammopiptanthus nanus Betaine aldehyde dehydrogenase Heat Heterologous expression Salt Tolerance
a b s t r a c t Betaine aldehyde dehydrogenase (BADH) catalyzes the synthesis of glycine betaine, a regulator of osmosis, and therefore BADH is considered to play a significant role in response of plants to abiotic stresses. Here, based on the conserved residues of the deduced amino acid sequences of the homologous BADH genes, we cloned the AnBADH gene from the xerophytic leguminous plant Ammopiptanthus nanus by using reverse transcription PCR and rapid amplification of cDNA ends. The full-length cDNA is 1868 bp long without intron, and contains an open reading frame of 1512 bp, and 3′- and 5′-untranslated regions of 294 and 62 bp. It encodes a 54.71 kDa protein of 503 amino acids. The deduced amino acid sequence shares high homology, conserved amino acid residues and sequence motifs crucial for the function with the BADHs in other leguminous species. The sequence of the open reading frame was used to construct a prokaryotic expression vector pET32a-AnBADH, and transform Escherichia coli. The transformants expressed the heterologous AnBADH gene under the induction of isopropyl β-D-thiogalactopyranoside, and demonstrated significant enhancement of salt and heat tolerance under the stress conditions of 700 mmol L−1 NaCl and 55 °C high temperature. This result suggests that the AnBADH gene might play a crucial role in adaption of A. nanus to the abiotic stresses, and have the potential to be applied to transgenic operations of commercially important crops for improvement of abiotic tolerance. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Drought, salinity, extreme temperatures and other abiotic stresses significantly restrict productivity and quality of crops (Hu and Xiong, 2014; Roy et al., 2014; Tambo and Abdoulaye, 2012), which adapt themselves to these abiotic stresses through a series of mechanisms (Dong et al., 2014; Neumann, 2008; Zheng et al., 2010). Compatible solutes, such as proline, glycine betaine and soluble sugars play a crucial role in the process counteracting stress via balancing osmotic potential (Chen and Murata, 2011; Dong et al., 2014; Silvente et al., 2012). Glycine betaine is an important osmoprotectant that can stabilize the structure and function of biomembrane system, enzymes, photosystem II complexes, ribulose-1,5-bisphosphate carboxylase/oxygenase, and many other functional proteins (Bao et al., 2011; Carillo et al., 2008, 2011; Chen and Murata, 2008; Gill et al., 2014; Prasad and Saradhi, 2004). In Abbreviations: RACE, rapid amplification of cDNA ends; BADH, betaine aldehyde dehydrogenase; ORF, open reading frame; CTAB, hexadecyltrimethylammonium bromide; UTR, untranslated region; IPTG, isopropyl β-D-thiogalactopyranoside; LB, Luria–Bertani; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. ⁎ Corresponding authors. E-mail addresses: ffl@sicau.edu.cn (F.-L. Fu), [email protected] (W.-C. Li). 1 Contributed equally.
http://dx.doi.org/10.1016/j.gene.2014.07.049 0378-1119/© 2014 Elsevier B.V. All rights reserved.
plants, the biosynthesis of glycine betaine consists of two steps: choline oxidation to betaine aldehyde catalyzed by choline monooxygenase, and betaine aldehyde to glycine betaine via deoxygenation catalyzed by betaine aldehyde dehydrogenase (BADH) (Rasheed et al., 2011). The heterologous overexpression of betaine aldehyde dehydrogenase genes increased betaine aldehyde accumulation, and improved tolerance of the transgenic plants to drought, salinity, extreme temperatures and other abiotic stresses (Fan et al., 2012; Karabudak et al., 2014; Li et al., 2011, 2014; Wu et al., 2008; Zhang et al., 2012; Zhou et al., 2008). However, the different improved phenotypes were obtained from these transgenic events. Except for the differentiation of gene expression, the activities of the BADHs encoded by the transformed exogenous genes themselves were probably one of the major reasons. Therefore, cloning and function evaluation of new betaine aldehyde dehydrogenase genes, particularly from abiotic stress-tolerant plant species, become helpful for transgenic operation of commercially important crops for improvement of abiotic tolerance. Ammopiptanthus nanus, one of the two relict species of the Ammopiptanthus genus (Leguminosae), is the unique evergreen broadleaf bush in the plateau desert from the west edge of the Tarim Basin to the border between China and Kyrgyzstan (Cheng, 1959). It is endemic to the harsh environments of arid climate (annual precipitation
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b 200 mm), extreme temperatures (from − 30 °C to 40 °C), dryness (dryness usually more than 4), poor soil quality and high salinity (the habitats are usually stony and/or sandy slopes) (Pan et al., 1992; Wang, 2005; Wang et al., 2007; Yan et al., 2000). According to the criteria that 80%–100% of leaves are severely affected (Miller, 1963), the critical high temperature of A. nanus is 65 °C, significantly higher than other sandy plants (Liu and Qiu, 1982; Liu et al., 1995). A germination test showed that the critical concentration of NaCl that permits the germination of A. nanus seeds was 1.38% (Wang and Yin, 1991). Another study of our research team has demonstrated that the antifreeze protein gene of A. nanus has stronger function than those cloned from other plants (Deng et al., 2014). Therefore, it is promising to clone and evaluate other stress-related genes from this tenacious species for understanding the molecular mechanism of tolerance to abiotic stress and probable application in transgenic operation. This paper reports the cloning of the AnBADH gene from A. nanus and the function evaluation by heterologous expression in Escherichia coli.
2. Materials and methods 2.1. Plant material and NaCl treatment The seeds of A. nanus were soaked in 60 °C water for 5 min, and sown in sterilized nutrient soil. The seedlings were grown at 26/30 °C under the light/dark cycle of 12/12 h for four weeks, and then subjected to salt stress by adding 500 mmol L − 1 NaCl, in order to increase the expression of the AnBADH gene. Five hours later, the leaves were harvested, snap frozen in liquid nitrogen and stored at − 80 °C.
2.2. Cloning of full-length cDNA Total RNA was extracted from the stored leaf sample by using RNAiso plus kit (TaKaRa, Japan), and treated with RNase-free DNase to remove probable genomic DNA. The first strand of the cDNA was reverse transcribed by using the total RNA sample as the template, and PrimeScript™ II 1st Strand cDNA Synthesis kit (TaKaRa, Japan). A pair of primers (5′-CTGTGAAT GGCGACACGGAAG-3′/5′-GATTGTTCCACGCTCGCTCTTAG-3′) was designed according to the partial cDNA sequence (GenBank accession number: DQ288723) of the BADH gene in Ammopiptanthus mongolicus, the other species of the Ammopiptanthus genus, and used to amplify the core sequence of the AnBADH gene with the sample of the first strand of the cDNA, and high fidelity DNA polymerase (TaKaRa, Japan). The first strand of the cDNA for 3′-RACE (rapid amplification of cDNA ends) was reverse transcribed by using the total RNA as the template, and the 3′-RACE adaptor with 3′-Full RACE Core Set Ver.2.0 (TaKaRa, Japan). The product was used for the first amplification of the nested PCR with the specific outer primer (5′-ACTGGAAGCTCTGCAACTGGGA CCAAGA-3′) designed according to the core sequence, and the 3′RACE outer primer (5′-TACCGTCGTTCCACTAGTGATTT-3′). The product was used for the second amplification of the nested PCR with the specific inner primer (5′-TCAAGCCTGTTTCACTAGAGCTCGGTGG-3′) designed according to the core sequence, and the 3′-RACE inner primer (5′-CGCG GATCCTCCACTAGTGATTTCACTATAGG-3′). The 5′-RACE was conducted using First Choice® RLM-RACE kit (Ambion, USA). The total RNA sample was dephosphorylated with calf intestinal alkaline phosphatase, decapped with tobacco acid pyrophosphatase, ligated with the 5′ RACE adaptor, and used for the reverse transcription of the first strand of the cDNA with the random
Fig. 1. Prokaryotic expression vector pET32-AnBADH. The AnBADH gene is under the control of the promoter and terminator of phage T7. The sequence immediately after the promoter encodes a leader peptide including a sulfoxide reductase protein tag (Trx-tag), two histidine selective tags (His-tag) and an S-tag for western blotting. The ampicillin resistance gene Ap is used as the selective marker.
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Fig. 2. Full-length cDNA sequence of AnBADH gene in A. nanus. The frame and asterisk indicate the start codon and the stop codon, respectively.
decamer primer. The product was used for the first amplification of the nested PCR with the specific outer primer (5′-GCACCAGCTTCA GGACCTAATCCAGTGA-3′) designed according to the core sequence, and the 5′-RACE outer primer (5′-GCTGATGGCGATGAATGAACAC TG-3′). The product was used for the second amplification of the nested PCR with the specific inner primer (5′-CCAGCTCCAAACAG GTCACAGATGCCAA-3′) designed according to the core sequence, and the 5′-RACE inner primer (5′-CGCGGATCCGAACACTGCGTTTGC TGGCTTTGATG-3′).
The amplified products of the core sequence, 3′-RACE, and the 5′-RACE were subcloned into the pMD19-T vector (TaKaRa, Japan), sequenced at Invitrogen (USA) and Sangon (China), repeatedly, and assembled into the full-length cDNA sequence. 2.3. Amplification of genomic DNA sequence Total DNA was extracted from stored leaf sample by the method of CTAB (Doyle and Doyle, 1987), and used to amplify the genomic DNA
Fig. 3. Phylogenetic tree among the putative and the BADH proteins in other plants.
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Fig. 4. Five crucial domains of AnBADH and four other BADH proteins. The asterisk, double and single-spot indicate perfect (100%), high (75%), and low (25%) conservation of the amino acids, respectively. The gray backgrounds, black triangle and solid circles indicate NAD (P)+ binding sites, catalytic residues, and the transit signal, respectively.
sequence of the AnBADH gene with a pair of specific primers (5′-ATGG CAA CCCCAATACCGAAT-3′ /5′-CCTATCACAGCTTTGAAGGAGACTG-3′) designed according to the full-length cDNA sequence, and Prime STAR HS DNA Polymerase (TaKaRa, Japan). The product was added with an adenosine triphosphoric acid to each of the 3′-ends by using Taq DNA Polymerase (Tiangen, China), subcloned into the pMD19-T vector, sequenced at Invitrogen (USA) and Sangon (China), repeatedly, and used for alignment with the full-length cDNA sequence.
ORF sequence by using Prime STAR HS DNA Polymerase (TaKaRa, Japan). The product was digested by BamH I/Hind III, and inserted into a prokaryotic expression vector pET32a (Fig. 1). The E. coli strain Rosetta (DE3) was transformed by the loaded expression vector pET32aAnBADH and empty prokaryotic pET32a (negative control), and screened on a Luria–Bertani (LB) agar plate containing 50 mg L− 1 ampicillin at 37 °C overnight. 2.6. Induced expression and stress treatment
2.4. Bioinformatics analysis The open reading frame (ORF) of the AnBADH gene was identified from the full-length cDNA sequence by online software ORF finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). The putative amino acid sequence was deduced online (http://web.expasy.org/translate/) from the full-length cDNA sequence of the AnBADH gene, and used for multiple alignment with BADHs of relative and model species by using ClustalX2.1 software (http://mac.softpedia.com/get/Math-Scientific/ ClustalX.shtml). The physical and chemical properties, secondary structure, transmembrane regions and orientation, subcellular location and signal peptide were predicted by using Expasy (http://www.expasy. org/), GOR IV (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page= npsa_gor4.html), TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/ services/TMHMM-2.0/), WoLF PSORT (http://wolfpsort.seq.cbrc.jp/), and SignalP 4.1 (http://www.cbs.dtu.dk/services/SignalP/), respectively. The phylogenetic tree was constructed by using software MEGA5.1 (http://mega.software.informer.com/5.1b/). The conserved domains were analyzed by using Conserved Domains Search Tool (http://www. ncbi.nlm.nih.gov/cdd/). 2.5. Transformation of E. coli A pair of specific primers (5′-GTGGATCCATGGCAACCCCAATACCG AAT-3′/5′-CATCAAGCTTCCTATCACAGCTTTGAAGGAGACTG-3′) was designed with the introduction of the recognition sites of BamH I and Hind III (the underlined bases in the bracket), and used to amplify the
After confirmation by bacterial PCR and double digestion of the extracted plasmids with BamH I/Hind III, the positive single colonies were cultured in LB liquid medium containing 50 mg L−1 ampicillin at 37 °C. After 5–6 h until OD600 ≈ 0.6, the cultures were added with isopropyl β-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mmol L−1, and incubated at 37 °C for 4 h. Then, each of the cultures was adjusted to OD600 ≈ 0.6 with the ampicillin LB liquid medium, and divided into five portions. From each of the first portions, the E. coli cells were harvested, resuspended in 1× PBS buffer and 2 × SDS sample buffer, lysed in a boiling water bath for 5 min, centrifuged at 13,000 r/min for 10 min, and used for the detection of the heterologous expression of the AnBADH gene by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Referring to He et al. (2012) and Udawat et al. (2014), each of the second portions was diluted by ten-fold serial to 1:105 with the ampicillin LB liquid medium. Five microliters of each of the dilutions was spotted onto the ampicillin LB agar plates containing NaCl of 170 (control), 300, 500, 700 and 1000 mmol L−1. After incubation at 37 °C for 16 h, the growth status of the colonies was observed, and used to determine the suitable NaCl concentration for high salt treatment. One milliliter was taken from each of the third portions, and added into 20 mL of the ampicillin LB liquid medium containing NaCl of the suitable concentration for high salt treatment. OD600 value was measured at every hour during incubation at 37 °C, and used for the analysis of the growth dynamics.
Fig. 5. Conserved domains of the putative protein. A NAD (P)+-dependent ALDH-SF and NAD (P)+ binding sites are included.
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Each of the fifth portions was subjected to heat treatment of 55 °C for the suitable duration, and diluted to 1:108 with the ampicillin LB liquid medium. One hundred microliters was taken from the dilutions, and spread onto the ampicillin LB agar plates with three replicates. After incubation at 37 °C for 16 h, colonies were counted, and used to calculate the average survivals of the three replicates. 3. Results 3.1. Full-length cDNA sequence The amplified core sequence was 585 bp in length, and shared high similarity to the BADH genes in other leguminous plants. 1046 bp and 654 bp fragments were amplified with the inner primers in the second amplification of the 3′-RACE and 5′-RACE, respectively. The overlapping regions of these three sequences were perfectly matched, and assembled into a full-length cDNA of 1868 bp long, with a 5′-untranslated region (UTR) of 62 bp, an open reading frame (ORF) of 1512 bp, and a 3′UTR of 294 bp (Fig. 2). The alignment with the genomic DNA sequence showed that there was no intron in the genomic DNA sequence. Fig. 6. Heterologous expression of AnBADH gene in E. coli fractionated by SDS-PAGE. Lane M: molecular weight marker; lane 1: uninduced transformant by empty vector pET32a; lane 2: IPTG-induced transformant by empty vector pET32a; lane 3: uninduced transformant by loaded vector pET32a-AnBADH; lane 4: IPTG-induced transformant by loaded vector pET32a-AnBADH, the biggest band is the fusion protein of the leader peptide and the AnBADH.
Referring to He et al. (2012) and Wu et al. (2012), each of the fourth portions was subjected to a heat treatment of 55 °C. At the 0th (control), 5th, 10th, 15th, 30th, 45th and 60th min, the cultures were sampled, and diluted by ten-fold serial to 1:105 with the ampicillin LB liquid medium. Five microliters of each of the dilutions was spotted onto the ampicillin LB agar plates. The growth status of the colonies was observed after incubation at 37 °C for 16 h, and used to determine the suitable duration of heat treatment.
3.2. Properties of putative protein The putative protein of the 1512 bp ORF contains 503 amino acids, with 54.71 kDa, isoelectric point pH 5.39, instability index (II) 28.36 (b40), and grand average of hydropathicity (GRAVY) − 0.015 (b0). The percentages of α-helices, ß-sheets and random coils in the secondary structure were predicted to be 41.4%, 7.6% and 34.19%, respectively. Its subcellular localization was predicted to be chloroplast. These properties suggest that the putative protein is a stable hydrophilic protein, neither a transmembrane protein nor a secreted protein. The multiple alignment showed that the amino acid sequence of the putative protein shares the highest identity with BADHs of leguminous plants, such as Glycine max (GenBank accession no. ADN03184.1) and Phaseolus vulgaris (GenBank accession no. XP_007138125.1) (Fig. 3). Particularly, the transit signal Ser-Lys-Leu (SKL) at the C-terminal (Fig. 4), the conserved domains
Fig. 7. Growth status of the E. coli colonies transformed by pET32a-AnBADH and pET32a under different salt concentrations.
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Fig. 8. Growth status of the colonies transformed by pET32a-AnBADH and pET32a after 55 °C heat treatment for different times.
of the nicotinamide adenine dinucleotide phosphate-dependent aldehyde dehydrogenase superfamily [NAD(P)+-dependent ALDH-SF] and the nicotinamide adenine dinucleotide phosphate [NAD (P)+] binding sites are essential for the function of BADH (Figs. 4 and 5) (Diaz-Sanchez et al., 2012; Reumann, 2004). All these properties identify the putative protein as a betaine aldehyde dehydrogenase, and its encoding sequence as the AnBADH gene. Therefore, we registered this sequence at GenBank with accession number KJ841914.
3.3. Heterologous expression of AnBADH gene confers high salt and heat tolerance to E. coli The result of SDS-PAGE revealed an obvious additional band about 76 kDa present in the IPTG-induced transformed line by the loaded vector pET32a-AnBADH, but absent in the other three negative controls, indicating the heterologous expression of the AnBADH gene in the transformed E. coli line (Fig. 6). Figs. 7 and 8 show the gradual decrease of the colony number on the LB agar plates along with the increase of the salt concentration, duration of the heat treatment, as well as the dilution fold. However, obvious differences were present between the lines transformed by the loaded vector pET32a-AnBADH and by the empty vector pET32a. The suitable NaCl concentration for high salt treatment is 700 mmol L−1, and the suitable duration of the 55 °C heat treatment is 15 min. Fig. 9 shows the growth dynamics of the transformed lines by the loaded vector pET32a-AnBADH and the empty vector pET32a. In the control medium (170 mmol L−1 NaCl), the growth speed of these two lines was not significantly differential, and entered the exponential growth phase in a short time. In the high salt medium (700 mmol L−1 NaCl), however, the growth of the transformed line by the loaded vector pET32a-AnBADH increased significantly faster than the transformed line by the empty vector pET32a. The range of this differentiation increased with time. As shown in Fig. 10, the colony number of the transformed line by the loaded vector pET32a-AnBADH on the ampicillin LB agar plates was obviously more than that of the transformed line by the empty vector pET32a. The average survival rates of the three replicates were 45.3% for the transformed lines by the loaded vector pET32a-AnBADH, and 29.8% for that by the empty vector pET32a. This differentiation was proven to be significant by t-test (p b 0.01). 4. Discussion
Fig. 9. Growth curves of the transformed lines by pET32a-AnBADH and pET32a in the control (170 mmol L−1 NaCl) and the high salt (700 mmol L−1 NaCl) mediums. 1: IPTGinduced transformant by loaded vector pET32a-AnBADH in the control mediums; 2: IPTG-induced transformant by empty vector pET32a in the control mediums; 3: IPTGinduced transformant by vector pET32a-AnBADH in the high salt mediums; 4: IPTGinduced transformant by empty vector pET32a in the high salt mediums.
There was a fusion gene on the pET32a vector that encodes a 15.3 kDa fusion leader peptide including a sulfoxide reductase protein tag (Trx), two histidine selective tags (His) and an S tag for western blotting (Fig. 1). Its overexpression induced by IPTG might cause metabolic burden and inhibit the expression of the endogenous proteins in E. coli. This was demonstrated by the weaker background bands in lanes 2 and 4 loaded with the IPTG-induced lysates than those in lanes 1 and 3
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Fig. 10. E. coli colonies transformed by pET32a-AnBADH and pET32a after 55 °C heat treatment for 15 min. I, II, III indicate three replicates, respectively.
loaded with the uninduced lysates in Fig. 6, and by the similar results in the transformed E. coli lines by other heterologous genes (Aris et al., 1998; Deng et al., 2014; Kurland and Dong, 1996; Li et al., 2013; Ricci and Hernandez, 2000). Therefore, we did not conduct IPTG induction in the incubation for high salt and heat treatment. The additional band in lane 3 loaded with the uninduced lysate, which displayed the same migration rate as the biggest band in lane 4 loaded with the IPTG-induced lysate, demonstrated the heterologous expression of the AnBADH gene without IPTG induction (Fig. 6). The results of the present study indicated that the heterologous expression of the AnBADH gene from A. nanus confers high salt and heat tolerance to E. coli, which has been sufficiently evaluated to be an effective heterologous expression system for plant genes (He et al., 2012; Lan et al., 2005; Li et al., 2013; Wu et al., 2012), and particularly for genes from the Ammopiptanthus genus (Deng et al., 2014; Liu et al., 2010). The heterologous expression of the BADH genes from other plants has been reported to increase the accumulation of glycine betaine, and enhance the tolerance of E. coli to abiotic stresses (Yang et al., 2012; Yilmaz and Bülow, 2002). The heterologous expression of the BADH genes from other plants also resulted in improvement of the transgenic plants for abiotic stresses (Fan et al., 2012; Karabudak et al., 2014; Li et al., 2011, 2014; Wu et al., 2008; Zhang et al., 2012; Zhou et al., 2008). Therefore, the results of the present study suggest that the expression of the AnBADH gene and the accumulation of glycine betaine are two of the molecular mechanisms of how A. nanus copes with the harsh environments. These mechanisms imply the potential of the AnBADH gene to be applied to genetic modification of commercially important crops for improvement of abiotic tolerance.
5. Conclusion The full-length cDNA of the AnBADH gene from A. nanus is 1868 bp long without intron, and contains an open reading frame of 1512 bp, encoding a 54.71 kDa protein of 503 amino acids. Its heterologous expression confers high salt and heat tolerance to E. coli, suggesting that the AnBADH gene might play a crucial role in adaption of A. nanus to the abiotic stresses, and have the potential to be applied to transgenic operations of commercially important crops for improvement of abiotic tolerance.
Conflict of interest There is no conflict of interest in the submission of this manuscript.
Acknowledgments This work was supported by the National Key Science and Technology Special Project (2013ZX08003-005), and the National Natural Science Foundation of China (31071433). The authors thank the technical support from the Key Laboratory of Biology and Genetic Improvement of Maize in Southwest Region, and the anonymous reviewers for their critical reading and modification suggestion.
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