Molecular cloning and characterization of the MsHSP17.7 gene from Medicago sativa L.

Heat shock proteins (HSPs) are ubiquitous protective proteins that play crucial roles in plant development and adaptation to stress, and the aim of this study is to characterize the HSP gene in alfalfa. Here we isolated a small heat shock protein gene (MsHSP17.7) from alfalfa by homology-based cloning. MsHSP17.7 contains a 477-bp open reading frame and encodes a protein of 17.70-kDa. The amino acid sequence shares high identity with MtHSP (93.98 %), PsHSP17.1 (83.13 %), GmHSP17.9 (74.10 %) and SlHSP17.6 (79.25 %). Phylogenetic analysis revealed that MsHSP17.7 belongs to the group of cytosolic class II small heat shock proteins (sHSP), and likely localizes to the cytoplasm. Quantitative RT-PCR indicated that MsHSP17.7 was induced by heat shock, high salinity, peroxide and drought stress. Prokaryotic expression indicated that the salt and peroxide tolerance of Escherichia coli was remarkably enhanced. Transgenic Arabidopsis plants overexpressing MsHSP17.7 exhibited increased root length of transgenic Arabidopsis lines under salt stress compared to the wild-type line. The malondialdehyde (MDA) levels in the transgenic lines were significantly lower than in wild-type, although proline levels were similar between transgenic and wild-type lines. MsHSP17.7 was induced by heat shock, high salinity, oxidative stress and drought stress. Overexpression analysis suggests that MsHSP17.7 might play a key role in response to high salinity stress. Electronic supplementary material The online version of this article (doi:10.1007/s11033-016-4008-9) contains supplementary material, which is available to authorized users.


Introduction
Plant HSPs normally participate in responses to drought, heat shock, salinity, heavy metals and peroxide stress [1]. HSPs act as molecular chaperones that bind other proteins to maintain steady-state target protein and promote the recovery of denatured proteins, which allows homeostasis of the internal environment during plant development and stress adaptation [2]. Small heat shock proteins (sHSPs, 15-42 kDa) form 200-800 kDa multimeric chaperone complexes [3] and are divided into six classes based on sequence similarity and cellular location. Class CI, CII and CIII sHSPs localize to the cytosol or nucleus [4], as well as the endoplasmic reticulum, mitochondria, and membranes [5]. sHSPs just bind to misfolded or denatured protein substrates, whereas refolding steps are mediated by Hsp70/ Hsp100 complexes [6], preventing irreversible unfolding or aberrant protein aggregation [7]. Their ability to prevent irreversible protein aggregation and to resolubilize aggregated proteins allows native proteins to remain in a stable state. Thus, sHSPs have been described as the 'paramedics of the cell' [8,9].
Several studies have shown that numerous plant sHSPs participate in the development of nutritive tissue, embryogenesis, germination and fruit production [10]. In addition, sHSP plays a significant role in the response to abiotic stresses. The Arabidopsis AtHsp15.7 gene is expressed at very low levels in a normal environment but is Electronic supplementary material The online version of this article (doi:10.1007/s11033-016-4008-9) contains supplementary material, which is available to authorized users. highly expressed upon heat shock or oxidative stress [11]. A previous study demonstrated that overexpression of sHSP17.7 increased drought tolerance in transgenic rice seedlings [7], and Lee et al. found that overexpression of the MsHSP23 gene enhanced salinity and arsenic tolerance in transgenic tobacco [3].
Alfalfa (Medicago sativa L.) is an important leguminous forage plant and is cultivated worldwide because of its high nutritional value and yield. Adverse external conditions, such as salinity, drought, high temperature and other types of stress, have a negative impact on the yield and quality of alfalfa. Therefore, improved stress resistance is a priority in breeding efforts to produce novel varieties of alfalfa that are better adapted to specific local environments and varying climate conditions. Here, we report the characterization and function of MsHSP17.7 gene from alfalfa, a possible candidate gene for stress tolerance in M. sativa.

Materials and methods
Plant materials and growth conditions M. sativa L. cv. Zhongmu No.1 and Arabidopsis thaliana (ecotype; Col-0) were used for gene cloning and genetic modifications. Thirty alfalfa seedlings per pot were cultured in a Hoagland hydroculture system in a plant growth chamber with 60 % humidity, a temperature of 24°C, and  Cloning of the MsHSP17.7 gene and bioinformatics analysis Homology-based cloning was performed to obtain the open reading frame (ORF) of MsHSP17.7. Plant total RNA was extracted from alfalfa using the MiniBEST plant RNA extraction kit (Takara Biotech Co., Ltd., Dalian, China), and cDNA was subsequently obtained using the Prime-Script RT-PCR Kit (Takara). MsHSP-F (sequence: 5 0 -CCTCCCATAATCTTCCAACCAC-3 0 ) was used as the sense primer, and MsHSP-R (sequence: 5 0 -CAAAAAAC CATTGCCACACACG-3 0 ) was used as the antisense primer. DNA fragment were cloned using alfalfa cDNA as the template by ordinary PCR. The obtained DNA fragment was then ligated into the pEASY-T1 vector, which was purified from positive Escherichia coli colonies containing the MsHSP17.7 gene and the insert was sequenced.

Expression analysis of MsHSP17.7
To investigate the expression pattern of MsHSP17.7 in alfalfa under heat shock, high salinity, oxidative stress and drought stress, 25-day-old alfalfa seedlings were treated over a 24-h period to induce heat shock (37°C), salt stress (200 mM NaCl), oxidative injury (15 mM H 2 O 2 ) or drought conditions (200 g l -1 polyethylene glycol (PEG) 6000) in Hoagland solution. Three alfalfa seedlings were randomly selected at 0, 2, 4, 8, 12 and 24 h, and total RNA was extracted from roots, stems and leaves. Subsquently cDNA was reversely transcribed using the PrimeScript RT-PCR Kit (Takara) described above. Then quantitative reverse-transcription PCR (RT-qPCR) were performed on roots, stems and leaves of alfalfa. The specific primers MsHSP17.7 qhsp-f (5 0 -CACCACATAATGGACCTCACAGAT-3 0 ) and qhsp-r (5 0 -TGATGTCACCTGATTTCAACCCTG-3 0 ) were used in assays. The alfalfa b-actin gene (GenBank: JQ028730.1) was used as an internal control with the primers qact-f (5 0 -CAAAAGATGGCAGATGCTGAGGAT-3 0 ) and qact-r (5 0 -CATGACACCAGTATGACGAGGTCG-3 0 ). Then quantitative reverse transcription PCR (RT-qPCR) reactions were performed on roots, stems and leaves. The RT-qPCR protocol was performed as specified by the manufacturer's instructions for the SYBR Primix Ex Tap II kit (Takara). The mean threshold cycle (Ct) was used as a reference value to calculate the level of each mRNA. Three biological replicates and per replicate contains three alfalfa seedlings were performed. The variance was subjected to a least significant difference (LSD) test using SAS software (version 9.13).
Expression of MsHSP17.7 in E. coli and salinity and oxidative stress survival assays The complete MsHSP17.7 ORF was amplified with forward primer EHSP-F (5 0 -ATGGATTTCAGGCTAATGGGTTT GG-3 0 ) and reverse primer EHSP-R (5 0 -TCAAGCAACCT TAACCTCAATAGTCT-3 0 ) and ligated into a prokaryotic expression vector (pEASY-E2, Novagen, USA) to generate the expression plasmid pEASY-E2/MsHSP17.7. Then, the plasmid was transformed into the E. coli strain Transetta DE3, which was cultured at 37°C with shaking at 200 rpm. During log phase, 1 mM IPTG was added to bacterium solution to induce protein expression. The bacterial suspension was harvested by centrifugation for 0, 3, 5, 7 and 9 h, and the fusion protein was separated by 12 % SDS-PAGE and Coomassie brilliant blue (CBB) staining.
Tolerance to salt and oxidative stresses were tested by growing bacteria in LB medium supplemented with
The transformed Arabidopsis seedlings were transplanted into pots under the conditions described above.
To identify transgenic A. thaliana, genomic PCR and RT-PCR analyses were performed comparing wild-type and transgenic lines. The primers pBI-F and pBI-R were used to amplify the MsHSP17.7 gene using the genomic gene in A. thaliana plants as a template for PCR. Then, At-act-F (5 0 -GAAGTCTTGTTCCAGCCCTCGTTTG-3 0 ) and At-act-R (5 0 -GAACCACCGATCCAGACACTGTACT-3 0 ) were used to amplify the A. thaliana actin 2 gene (GenBank: NM_112764.3) based the cDNA template as a control. Additionally, pBI-F and pBI-R were used to amplify MsHSP17.7 from the cDNA template. In this experiment, T3 transgenic Arabidopsis homozygous lines, T31 and T37, were randomly selected and used throughout the study.

Analysis of transgenic Arabidopsis under stress conditions
For salt stress treatment, 15 seeds were randomly taken from T3 transgenic lines and wild-type A. thaliana. Arabidopsis were germinated on 1/2 MS medium containing 150 mM NaCl for 12 days, and the lengths of their roots were measured. As a control, 4-week wild-type Arabidopsis, then NaCl was added to the Hoagland solution to obtain a final concentration of 200 mM NaCl. At 36 h, the MDA and proline contents were measured. The MDA content was determined using the thiobarbituric acid (TBA) reaction, as described by Heath and Packer (1968). Three technical replicates and per replicate contains three Arabidopsis were performed. The variance was subjected to a LSD test using SAS software (version 9.13).

Results
Cloning and molecular characterization of MsHSP17.7 A 686 bp cDNA fragment containing a 477 bp ORF was amplified from alfalfa by homology cloning and designated b Fig. 2 Neighbor-joining phylogenetic tree of MsHSP17.7 and sHSPs from other plant species. The phylogenetic tree was constructed based on similarities with 50 plant sHSPs, which divided the sHSP gene family into six clades. Amino acid sequences used in the analysis were retrieved from GenBank or EMBL. Their database accession numbers are as follows:  Fig. 2B). In addition, the MsHSP17.7 protein was predicted to encode no transmembrane structures by TMHMM ( Supplementary Fig. 2C) Fig. 1. MsHSP17.7 shares high protein sequence identity with MtHSP (93.98 %), PsHSP17.1 (83.13 %), GmHSP17.9 (74.10 %) and SlHSP17.6 (79.25 %), and it shares higher sequence similarity with dicotyledons compared with monocotyledons. Based on a comparison with cytosolic class II sHSP sequences, a unique domain (RDAKAMAATPADV) was found in the N terminus (Fig. 1). A conserved C-terminal domain (a-Crystallin domain, ACD) of approximately 90 amino acids contained consensus regions II and III. Additionally, a polyproline motif PPPEPKKP was identified at the C-terminus [15][16][17]. A phylogenetic tree showed that MsHSP17.7 was identified as a member of the plant cytosolic class II sHSPs (Fig. 2).

Subcellular localization of the MsHSP17.7
GFP or the MsHSP17.7-GFP fusion protein was transiently expressed in onion epidermal cells. As shown in Fig. 3, the MsHSP17.7-GFP fusion protein accumulated mainly in the cytoplasm, whereas GFP alone was distributed throughout the entire cell. In addition to, the plasmolysis of onion cells indicated that MsHSP17.7-GFP fluorescence accumulated mainly in the cytoplasm instead of cell wall. This result was consistent with the prediction that MsHSP17.7 by ProtComp online.

Expression of MsHSP17.7 in alfalfa
The relative expression levels of MsHSP17.7 mRNA under different stress conditions are shown in Figs. 4, 5, 6 and 7. Under heat shock stress (Fig. 4), the pattern of expression in stems and leaves was consistent with that in roots. After a rapid increase after 2 h, the expression of mRNA MsHSP17.7 decreased as treatment time increased. At 2 h,  the level of MsHSP17.7 mRNA in the above-ground parts was 180.7-fold higher than in the control, and the expression in the underground parts was 436.5-fold higher than in the control. As shown in Fig. 5, the mRNA expression levels were higher under salt stress in the stems and leaves of stressed plants compared to those of control plants, except after 4 h of stress induction. However, there were no significant differences at 2, 4 and 12 h. The mRNA expression level was 4-fold higher in the above-ground tissues of stressed plants than in those of the control plants at 8 h. The expression level was the highest at 24 h (7-fold higher). The mRNA expression level of MsHSP17.7 in the root tissue gradually increased with treatment time until 12 h, at which time the level was 25-times higher than in the roots of the control plants. The mRNA expression level in the root suddenly decreased at 24 h of treatment to a level that was 5-fold higher than that of the control, whereas there was no obvious difference between expression levels at 0 and 24 h (Fig. 5B).
Upon peroxide stress treatment (Fig. 6A), the expression levels of MsHSP17.7 mRNA in the stems and leaves corresponded to those of the control at 4, 8 and 12 h. The expression levels at 2 and 24 h were slightly lower than those of the control at 0 h. The MsHSP17.7 mRNA expression pattern in alfalfa root was completely different, increasing 1.5-, 12.0-, 9.5-, 14.2-and 1.6-fold at 2, 4, 8, 12, and 24 h (Fig. 6B).
The expression pattern of MsHSP17.7 in stems and leaves was consistent with that in roots under osmotic stress (Fig. 7A, B). MsHSP17.7 expression levels in the whole plant were not significantly different from those of the control from 0 to 8 h. After 8 h, the expression levels  Expression of MsHSP17.7 in E. coli and salinity and oxidative stress survival assays Under the T7 promoter, the MsHSP17.7 protein was abundantly expressed in E. coli (Fig. 8), and SDS-PAGE analysis clearly showed a 17.70 kDa band. With the increased time of induction, the amount of IPTG-induced MsHSP17.7 expression was gradually increased (Fig. 8).
To evaluate whether MsHSP17.7 expression increased the capacity to resist salinity or peroxide stress in model microorganisms, the effects of MsHSP17.7 expression on the salinity and peroxide tolerance of E. coli were determined. In a controlled trial, as shown in Fig. 9A, there was no apparent difference in cell survival between the MsHSP17.7-expressing strain and the E2-expressing strain except at 2 and 4 h. However, the MsHSP17.7-expressing strain showed increased (P \ 0.05) survival after treatment with 200 mM NaCl compared to the vector control throughout the entire treatment period (Fig. 9B). Similarly, the MsHSP17.7-expressing strain showed tolerance to 15 mM H 2 O 2 treatment (Fig. 9C).

Analysis of transgenic Arabidopsis under stress conditions
In the transgenic lines L31 and L37 under normal growth conditions, the root lengths were 1.99 and 2.04 cm, respectively, as shown in Fig. 10, which were not significantly different from the wild-type line. Under salt stress, the root lengths of transgenic Arabidopsis lines were 1.5 and 1.53 cm, which were 0.3 and 0.33 cm longer than the wild-type line (P \ 0.01).
Under control conditions, the MsHSP17.7-overexpressing lines did not show significant differences in proline or MDA levels compared with wild-type Arabidopsis (Fig. 11). However, after treatment with 200 mM NaCl, the MDA content of the L31 and L37 lines were 12.2 and 13.0 nmol g -1 , respectively, and were significantly lower that of the wild-type line (Fig. 11A). There was no significant difference in the proline level between the transgenic and wild-type lines (Fig. 11B).

Discussion
Strategies employed by higher plants for surviving adverse conditions include behavioral adaptations, morphological changes and physiological regulation [18]. The role of sHSPs in protecting cells against damage related to abiotic and biotic stresses has been well demonstrated in organisms ranging from fungi [19,20] to plants [16,21]. The functions of sHSPs include binding to unfolded proteins and regulating their intracellular distribution, protein degradation and signal transduction, allowing cell survival under stress conditions [9]. In spite of the considerable research on the role of sHSP in response to environmental stresses in diverse plants, few studies on alfalfa sHSP have been reported. We cloned and characterized the MsHSP17.7 gene from alfalfa. A phylogenetic tree analysis divided 50 sHSPs into six families, of which Classes CI, CII and CIII are localized in the cytosol or nucleus [4].
Additionally, subcellular localization studies demonstrated that MsHSP17.7 is located in the cytosol. Combined with the evolutionary tree analysis, we propose that MsHSP17.7 is a cytosolic Class II sHSP.
sHSP genes are highly induced under conditions ranging from abiotic exposure to biotic stresses. It has been reported that A. thaliana CI sHSPs (Hsp17.4CI, Hsp17.6ACI, Hsp17.6BCI, and Hsp17.6CCI) are expressed during heat stress, osmotic stress, oxidative stress, UV-B exposure and other abiotic stressors [9]. HSPs are induced by heat, cold, drought, and oxidative and salt stresses in Oryza sativa [22]. In addition, Neta-Sharir demonstrated that tomato chloroplast sHSP21 is induced by heat treatment in leaves [23]. In this paper, the abundantly increased MsHSP17.7 mRNA indicated adaptation of the plant to the adverse environments. sHSP genes also respond to osmotic and salt stress. Ruibal et al. [24] reported that PpHsp16.4 was upregulated after exposure to various abiotic stress factors, including strong light, heat, salt and osmotic stress. In this study, MsHSP17.7 mRNA was up-regulated by NaCl and PEG treatments, suggesting that MsHSP17.7 might play a key role in osmotic and salt stress. AtHSP15.7, a peroxisomal sHSP, has been shown to be strongly induced by both oxidative and heat stress [11]. However, the analysis of an Arabidopsis cytosolic class II sHSP, AtHSP17.6A, revealed that this sHSP was induced by osmotic but not oxidative stress [25]. In our study, MsHSP17.7 mRNA expression was regulated in alfalfa roots under oxidative stress, but its levels remained stable in the stems and leaves. The At-HSP17.6A protein was not detected in PEGtreated Arabidopsis plants, whereas At-HSP17.6A mRNA was induced [15]. In our study, MsHSP17.7 was induced after chronic drought treatment. Therefore, MsHSP17.7 was presumed to be involved in the drought response and to play a crucial role in plant stress tolerance. Under high temperature, drought, high salt concentration, or exposure to various pathogens [26], the primary function of this sHSP is to promote refolding of non-native proteins that have been denatured under stress condition.
Previous studies have demonstrated that the constitutive overexpression of sHSPs in plants is associated with enhanced resistance to abiotic stress [15,16]. In this study, MsHSP17.7-expressing E. coli showed increased survival following salt stress and peroxide stress, respectively, compared with controls. In Arabidopsis, overexpression of wheat chloroplastic sHSP26 results in improved heat tolerance [27]. In this study, the transgenic Arabidopsis seedlings exhibited significantly longer root, lower MDA content and similar proline content compared to wild-type Arabidopsis under high salinity stress. The lower MDA content indicated that less damage occurred in the plant. Some researchers argue that proline is a compatible osmolyte that accumulates in plant cells in response to salt stress [28], but others favor the view that proline is simply a stress-induced product [29]. In the present study, there was no marked difference in the proline content between transgenic and wild-type Arabidopsis.
In conclusion, we showed that MsHSP17.7 is localized in the cytoplasm and is induced by heat shock, high salinity, peroxide and drought stress. Expression of MsHSP17.7 in transgenic E. coli and A. thaliana indicated that it could enhance salt tolerance.