ФИЗИОЛОГИЯ РАСТЕНИЙ, 2013, том 60, № 3, с. 369-376
ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ
УДК 581.1
Heterologous Expression of a Halophilic Archaeon Manganese Superoxide Dismutase Enhances Salt Tolerance in Transgenic Rice1 © 2013 Z. Chen****, Y. H. Pan**, L. Y. An*, W. J. Yang*, L. G. Xu*, C. Zhu***
* College of Life Sciences, Zhejiang University, Hangzhou, P.R. China ** College of Life Sciences, China Jiliang University, Hangzhou, P.R. China *** School of Life Sciences, Taizhou University, Taizhou, P.R. China Received May 22, 2012
In order to investigate new gene resource for enhancing rice tolerance to salt stress, manganese superoxide dismutase gene from halophilic archaeon (Natrinema altunense sp.), (NaMnSOD), was isolated and introduced into Oryza sativa L. cv. Nipponbare by Agrobacterium-mediated transformation. The transformants (L1 and L2) showed some NaMnSOD expression and increased total SOD and CAT activity, which contributed to higher efficiency of ROS elimination under salt stress. The levels of superoxide anion radicals (O2 ) and hydrogen peroxide (H2O2) were significantly decreased. In addition, they exhibited higher levels of photosynthesis, whereas lower relative ion leakage and MDA content compared to wild-type plants. Therefore, transgenic seedlings were phenotypically healthier, and heterologous expression of NaMnSOD could improve rice salt tolerance.
Keywords: Natrinema altunense sp. - Oryza sativa - superoxide dismutase - salt stress - transgenic rice
DOI: 10.7868/S0015330313030056
INTRODUCTION
There are many extremely harsh environments, such as hot springs, salt lakes, and submarine volcanic habitats with abundant resources of "extremophiles". The abnormal temperature, low nutrient levels, abundant sunlight, and remote geographical location of these regions make them relatively special ecosystems [1, 2]. However, isolation and applications of stress genes in extremophiles still remains quite limited. With the excess consumption of resources and the deterioration of the environment, characterization of gene information and protein properties of extremo-philes, as well as expression in other organisms promise to be beneficial for tolerance.
Salinity is a global problem that threatens crop yield and quality [3, 4]. Exposure to severe salt stress
1 This text was submitted by the authors in English.
Abbreviations'. CaMV - cauliflower mosaic virus; CAT - catalase; Chl - chlorophyll; F0 - the minimum fluorescence yield; Fm -the maximum uorescence yield; Fs - stable fluorescence yield; GUS - P-glucuronidase; L1/L2.- transgenic lines; NBT - ni-troblue tetrazolium chloride; O2 - superoxide anion radical; PCR - polymerase chain reaction; RT-PCR - reverse transcrip-tion-polymerase chain reaction; SOD - superoxide dismutase; WT - wild type.
Corresponding author. C. Zhu. College of Life Sciences, Zhejiang University, Hangzhou 310058, P.R. China. E-mail. pzhch@cjlu.edu.cn
can lead to oxidative damages, ion toxicity, and nutritious imbalance [5]. ROS play an important role in these toxic effects [6]. In the antioxidative process, superoxide dismutase (SOD), as the first defense line,
converts superoxide radical (O2-) into hydrogen peroxide (H2O2). H2O2 can be rapidly decomposed into nontoxic components O2 and H2O by peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), or in the ascorbate-glutathione cycle [7]. Overexpression of SOD was in positive correlation with stress tolerance of many transgenic plants [8-10].
SOD can be classified into four groups. copper-zinc SOD (Cu/ZnSOD), iron SOD (FeSOD), manganese SOD (MnSOD), and nickel SOD (NiSOD). MnSOD could be found in prokaryotic organisms as well as in eukaryotes. NaSOD, putative MnSOD from halophilic archaeon (Natrinema altunense sp.) was isolated in our previous work [11]. By the experimental verification, the NaSOD was expressed in colibacillus, and the tolerance of the colibacillus to a high-salt environment enhanced. Up to now, overexpression of archaeon SOD in plants has not been reported. Rice (Oryza sativa) is a tremendously important food crop and is particularly sensitive to salt stress.
In this study, NaSOD was introduced into rice by Agrobacterium-mediated transformation and proved to be a MnSOD coding gene. Phenotypic changes and
physiological parameters were measured, and the results showed the transgenic rice plants were more tolerant to salt stress than wild-type (WT) plants.
MATERIALS AND METHODS
Construction of vector and rice transformation. An
extremely halophilic archaeon strain (Natrinema al-tunense sp., AJ2) was isolated from Ayakekum salt lake in Altun Moutain in Xinjiang, China [1]. For protein screening, AJ2 was cultured with 1.7 M or 3.0 M NaCl and harvested in the logarithmic growth phase. Differential proteins were analyzed and purified with SDS-PAGE and HPLC. A DNA fragment of NaSOD was cloned after amino acid sequencing and PCR-ampli-fication. Homologous analysis of sequences and amino acid similarity indicated that the NaSOD was a MnSOD.
For construction of the plant express vector, the primers NaMnSOD-F, 5'-CGG GGT ACC ATG ACT GAT CAC GAA CTT CCAC-3', and NaMnSOD-R, 5'-AAA CTG CAG TTA CTC GAA GTG GTC GAG GCAG-3' were used for fidelity PCR. Then subcloned into reconstructive vector pCAMBIA1301 under cauliflower mosaic virus 35S (CaMV 35S) promoter. The p1301-35S::NaMnSOD was ready for transforming rice after introducing into Agrobacterium tumefaciens strain EHA105.
Transformation of rice (Oryza sativa L., cv. Nip-ponbare) was achieved by co-cultivation of rice calli derived from mature seed scutella with A. tumefaciens containing p\30l-35S::NaMnSOD according to a previously reported protocol [12].
Screening of transgenic plants. All the putative T0 transgenic plants were screened using PCR analysis and GUS staining [13]. Plants of T1 generation were firstly screened by 50 mg/L hygromycin selection. The lines with 3 : 1 segregation ratio were chosen and propagated.
SOD activity staining was performed according to the method of Luo and Wang [14]. The total proteins (50 ^g per sample) were separated on a nondenaturing 10% PAGE with a 2.5% stacking gel and stained by ri-boflavin-nitroblue tetrazolium method for SOD activity. Incubation of the gels with 5 mM H2O2 inhibited FeSOD and Cu/ZnSOD to identify MnSOD isoforms in the transgenic and untransformed plants [15].
Growth condition and stress treatment. Seeds of homozygous lines, named as L1 and L2, and wild type (WT) rice were sterilized with 10% NaClO (v/v) for 20 min, rinsed three times with sterilized water, and soaked for 1-2 days at 37°C in the dark. Then germinated seeds were hydroponics with the Yoshida's culture solution [16] refreshed every 5 days. The seedlings were grown at a 13-h photoperiod (200 ^mol/(m2 s)), 26/22°C (day/night) temperature, and 80% relative humidity.
Uniform 35-day-old seedlings were transferred to culture solution containing 100 mM NaCl, while others without NaCl served as controls. After exposure to salt stress for 3 and 6 days, the roots and the shoots were collected separately for further assays.
Reverse transcription-polymerase chain reaction (RT-PCR). For the analysis of NaMnSOD expression level, 0.1 g leaves from WT and transgenic plants treated with 100 mM NaCl were collected. Total RNA was isolated using TRIzol reagent ("Invitrogen"). RT-PCR was performed using a Prime Script™ 1st Strand PCR Kit ("TaKaRa Biotech."). Reverse transcription products (2 ^L) were subjected to PCR with NaMnSOD primers. Actin gene was used as an internal control [17].
Determination of ion leakage and lipid peroxidation.
Leaf relative electrolyte leakage was detected with the methods described by Arora et al. [18] with some mod-ications. Leaf samples of 0.1 g from WT and transgenic plants were vacuum infiltrated in 20 mL of distilled water for 10 min and then left overnight at 25 °C. After overnight incubation, the initial electric conductivity (C1) was measured using a conductivity meter ("HORIBA"). Thereafter, samples were boiled for 10 min and total electric conductivity (C2) was read at 25°C. Relative electrolyte leakage was expressed as a percentage of the total conductivity: C1/C2 x 100%.
MDA was extracted from the leaves with 10% (w/v) trichloroacetic acid and determined according to Huang et al. [19]. MDA was expressed as nmol/g initial fresh weight.
Assay of superoxide anion radical and hydrogen peroxide. O2 were estimated by the method of monitoring the nitrite formation from hydroxylamine in the presence of superoxide radical [20]. H2O2 level was measured according to the previously described method [21] and calculated using the extinction coefficient of 0.28 ^mol/(M cm).
Enzyme assays. Enzymes were extracted according to the reported method [19]. Leaf tissue (0.1 g) was ground into powder in liquid nitrogen and homogenized in 2 mL of precooled (4°C) extraction buffer (50 mM potassium phosphate, pH 7.0, 1 mM EDTA, 3 mM DTT, and 5% PVP-40). The homogenate was centrifuged at 15000 g for 20 min at 4°C, and the resulting supernatant was collected for further antioxi-dant enzyme assays.
Total SOD activity was assayed by measuring its ability to inhibit the photochemical reduction of ni-troblue tetrazolium chloride (NBT) at 560 nm. The reaction mixture contained 50 mM potassium phosphate (pH 7.8), 0.1 mM EDTA, 67 ^M NBT, 13 mM L-methionine, 1.3 ^M riboflavin, and suitable aliquot of the enzyme extract. Reaction was carried out at 30°C, under light intensity of about 150 ^mol/(m2 s) through 10 min.
CAT activity was assayed by measuring the initial rate of H2O2 disappearance at 240 nm (extinction coefficient 39.4 mmol/(M cm)). The reaction mixture (3 mL) contained 50 mM PBS (pH 7.0), 0.1 mM EDTA, 2 mM H2O2, and the enzyme extract.
Measurements of photosynthetic gas exchange and chlorophyll fluorescence. Gas exchange of the second fully developed leaf was measured using an LI-6400 portable photosynthesis system ("LI-COR",
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