ФИЗИОЛОГИЯ РАСТЕНИЙ, 2013, том 60, № 3, с. 338-344
ЭКСПЕРИМЕНТАЛЬНЫЕ ^^^^^^^^^^^^ СТАТЬИ
Cloning and Characterization of a Plasma Membrane Na+/H+ Antiporter Gene
from Cucumis sativus1 © 2013 S. Wang*2, Z. Li**2, R. Rui***2, G. S. Fan*2, K. W. Lin*
*College of Landscape Architecture, Southwest Forestry University, Kunming, China; **College of Horticulture, Northwest A&F University, Yangling, Shanxi, China ***College of Civil Engineering, Southwest Forestry University, Kunming, China
Received May 16, 2012
A plasma membrane Na+/H+ antiporter gene (CsSOSl) was separated from cucumber (Cucumis sativus L.) plants by RT-PCR and RACE methods. Sequence analysis indicated that the full-length CsSOSl cDNA was 3638 bp long with an open reading frame of 3435 bp long encoding a protein of 1145 amino acids. The deduced protein contained conserved structural domains and shared a high similarity with plasma membrane type Na+/H+ antiporters from other plants. TMpred prediction showed that CsSOSl had 11 transmembrane domains. As shown by RT-PCR, the expression of CsSOSl was tissue-specific and increased in the root but decreased in the leaves with increasing NaCl concentration. In addition, expression of CsSOSl in ATX3 mutant yeast could grow on medium containing NaCl and enhanced AXT3 salt tolerance. These results suggest that the CsSOSl plays a key role in cucumber plants under salt stress.
Keywords: Cucumis sativus - plasma membrane Na+/H+ antiporter gene (SOSl) - cDNA cloning - yeast
With the development of greenhouse and big-arch shelter, secondary soil salinization resulted from inefficient rotation, irrigation, and excessive applying fertilizer becomes one of the major factors limiting agricultural production in China. Sodium is one of the major toxic ions in saline soils harmful to plants. To cope with salt stress, plants have evolved a variety of adaptation mechanisms . Na+ extrusion and Na+ compartmentation are two important mechanisms for plants. Compartmentation of Na+ into vacuoles could be accomplished by the action of the Na+/H+ antiporter in the vacuolar membranes. Meanwhile, the function of the Na+/H+ antiporter in the plasma membrane (NHA) is to extrude Na+ from the cells.
The first plasma membrane higher plant NHA gene was cloned fromArabidopsis . Subsequently, a series of plasma membrane Na+/H+ antiporter coding genes have been identified and cloned, such as OsNHAl and
1 This text was submitted by the authors in English.
2 These authors contributed equally to this work.
Abbreviations: NHA - Na+/H+ antiporter; RACE - Rapid Amplification of cDNA Ends.
Corresponding author: G. S. Fan. College of Landscape Architecture, Southwest Forestry University, Kunming 650224, China. Fax: +86-0871-386-3023; e-mail: firstname.lastname@example.org
OsSOSl from rice [3, 4], CnSOSl from sea grass , PhaNHAl from reed plants, TaSOSl from wheat, LgSOSl from salty grass (Limonium gmelinii), LeSOSl from tomato. Moreover, the overexpression of SOSl in transgenic plants could significantly improve salt tolerance of Arabidopsis . However, limited information is available about horticultural plants, particularly cucumber plants.
Cucumber is an important horticultural crop and mainly cultivated in greenhouse where secondary soil salinization threatens to become, or already is, a problem in China. Some species of cucumber show strong salt tolerance. More studies should be done to evaluate the key genes involved in cucumber salt tolerance. In this study, therefore, we cloned the new plasma membrane Na+/H+ antiporter from cucumber, analyzed its sequence characterization, expression in mutant yeast, and expression patterns under salt stress. This work may pave the way for improving the cucumber salt tolerance strategies by means of genetic engineering.
MATERIALS AND METHODS
Plant material. Seeds ofcucumber (Cucumis sativus L.) cultivar Shenlv72 obtained from Shanghai Jiao Tong University were germinated and grown in perlite in plant chamber (16-h photoperiod, 28°C). When the first pair of cucumber euphylla was fully expanded for
salt treatments, the cucumber seedlings were randomly selected and then grown in 0.5-strength Hoagland solutions containing 0, 25, 50, or 100 mM NaCl for 12 h. Roots, shoots, and leaves were harvested, frozen in liquid nitrogen, and then stored at -80 °C until RNA extraction.
Total RNA was isolated using Trizol Reagent ("In-vitrogen", United States) from the roots of cucumber seedlings. First-strand cDNA synthesis and RT-PCR were performed using a one-step RNA PCR kit ("Takara"). A degenerate sense primer, CsF1 (5'-AAA GYT TRC AYC AYT TCT GGG A-3') and an antisense primer, CsR1 (5'-AGC ATT YCC CAG TAW GCW GCY TG-3'), were designed based on the conserved regions of the plasma membrane Na+/H+ antiporter amino acid sequences from other plants. PCR amplification was performed for 35 cycles (94 ° C for 30 s, 58 ° C for 30 s, and 72 ° C for 1 min) followed by a final extension step of 8 min at 72°C. A fragment of 700 bp was amplified from the root cDNA. This fragment shared high similarity with the sequences of other plasma membrane antiporter genes with the blasting in GenBank.
Full-length cDNAs were obtained using 3' and 5' RACE (Rapid Amplification of cDNA Ends) approach as described previously . 3' RACE and 5' RACE amplifications were carried out using the gene-specific primers CsF3' (5'-CAC CAT TCA AAA GTC GCA GAC G-3') for 3' RACE and CsF5' (5'-CAG CGA TAA CAA CTC CAC TCA A-3'), respectively. A nested-PCR was then performed with a nested universal primer (NUP, provided in the kit) as the reverse primers. The PCR conditions were as follows: dena-turation of the cDNA at 94°C for 5 min, 35 cycles of amplification (94°C for 45 s, 56/58°C for 45 s, 72°C for 180/90 s), and final extension at 72°C for 10 min. About 2000-bp of 3' and 1000-bp of 5' sequences were obtained; then all amplified fragments were sequenced and assembled to deduce the full length of the CsSOS1 cDNA by vector NTI 11.5 software. Finally, the full-length cDNA of CsSOS1 was amplified with the specific primers CsF2 (5'-AAC GCA CGA ACA GTC TCA-3') and CsR2 (5'-CAA CCC TCT ATC AAA CAA-3'), using the following conditions: denaturation of the cDNA at 94°C for 5 min, 35 cycles (94°C for 30 s, 57°C for 30 s, 72°C for 5 min), and an extension step of 10 min at 72°C.
Expression of CsSOS1 in yeast. The yeast expression vectors pFL61 containing CsSOS1 were introduced into the AXT3 using an electro-transformation protocol. This AXT3 strain lacks the original Na+/H+ antiporter in the plasma membrane (NHA1) and the vacuolar membrane (NHX1) and also lacks the P-type ATPase involved in Na+ exclusion (ENA1-4) .
For growth tests, the yeast cells were grown on agar medium and in liquid YPG medium containing 100 mM NaCl. According to the protocol , saturated yeast cultures were diluted to an absorbance of 0.8 at 600 nm with YPG solution. Aliquots (3 ^L) aliquots of the OD 600 = 0.8 yeast cultures or tenfold serial dilutions were spotted onto different YPG plates and grown at 30°C for 48 h. Further, 3 ^L of OD 600 = 0.8 culture was inoculated into 50 mL of YPG medium with 150 mM NaCl. Strain growth was measured by reading the absorbance at 600 nm every four hours.
Expression analysis of CsSOS1 under salt stress. Similar size seedlings were treated with 0.5-strength Hoagland solutions containing 0, 25, 50, or 100 mM NaCl for 12 h. Total RNAs separately extracted from the roots, stems, and leaves of plants were reverse transcribed as described earlier. We used the RT-PCR technique to examine the CsSOS1 gene expression. Amplification reactions were carried out using the gene-specific primers CMF3 (5'-AAA AGC ACT TCC TAA TGA GAG-3') and CMR3 (5'-CGY GGC CGT CTC TAT GAR TA-3'). As an endogenous control, we used the gene-specific primers: ACTF (5'-TCA ATG TGC CTG CTA TGT ATG T-3') and ACTR (5'-CAT ACC GAT GAG AG ATG GCT G-3') for cucumber Actin3 gene (DQ115883), the house-keeping gene in cucumber described previously . The RT-PCR conditions were as follows: 94°C for 3 min,
27 cycles (94°C for 30 s, 58°C for 30 s, and 72°C for 1 min) and 72°C for 8 min. The PCR products (6 ^L) were separated on 1% (w/v) agarose gels, stained with ethidium bromide (10 mg/mL), and the quantity of products was analyzed with the gene analysis software package ("Gene Company").
RESULTS AND DISCUSSION
Characterization of CsSOS1 cDNA and sequence analysis
We isolated cDNA of CsSOS1 (accession no. JQ655747) using the RT-PCR and RACE methods. Sequence analysis (fig. 1) indicated that the full-length CsSOS1 cDNA was 3638 bp long with an open reading frame of 3435 bp long encoding a protein of 1145 amino acids with a theoretical molecular mass of
28 kD and a theoretical pi of 4.86. CsSOS1 protein shared a high similarity with the plasma membrane type Na+/H+ antiporter, 58% with rice OsSOS1 , and 64% identity with Arabidopsis SOS1 . However, CsSOS1 shows considerably less homology with the vacuolar Na+/H+ antiporter AtSOS1 (9.05%) in Arabidopsis and OsSOS1 (10.00%) in rice [11, 12]. A hydropathy plot (fig. 2) generated using the TMpred program (http:// www.ch.embnet.org/software/TMPRED_for m.html) indicated that CsSOS1 consisted of 11 putative hydro-phobic regions with a long hydrophilic tail in the C-ter-
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