ФИЗИОЛОГИЯ РАСТЕНИЙ, 2014, том 61, № 5, с. 698- 704
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СТАТЬИ
УДК 581.1
ISOLATION AND EXPRESSION ANALYSIS OF Cu/Zn-SUPEROXIDE
DISMUTASE GENES FROM THREE Caragana SPECIES1 © 2014 W. B. Zhang*, N. N. Li**, F. Qi*, X. Chen**, J. Hao**, C. R. Yu**, X. F. Lin**
*College of Forestry, Inner Mongolia Agricultural University, Hohhot, China **College of Life Sciences, Inner Mongolia University, Hohhot, China Received December 26, 2013
Caragana is a native desert shrub with high forage values and stress tolerance, as well as sand-fixing capabilities. Some Caragana species, including C. korshinskii, C. microphylla, and C. intermedia, are commonly used for vegetation restoration programs in the Loess Plateau region of northwestern China and are known to have ecological benefits and high commercial value. In this study, full-length sequences of Cu/Zn-SOD genes were isolated from three Caragana species using degenerate polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE) techniques, and their expression under drought stress conditions were investigated. The cloned SOD cDNAs contained a predicted open reading frame of 459 bp encoding a polypeptide of 152 amino acids with a theoretical molecular weight of 15.2 kD. Cu/Zn-SOD cDNA of C. korshinskii and C. intermedia shared 100% sequence identity, implying a close relationship. A 24-bp specific sequence was found in the 3-UTR region of C. microphylla Cu/Zn SOD-cDNA, and reverse transcription RT-PCR and genomic PCR confirmed the feasibility of the 24-bp sequence as a DNA marker for rapid variety identification of C. microphylla. RT-PCR revealed that the expression of the Caragana Cu/Zn-SOD genes was induced by PEG-simulated drought stress and ABA. The three Caragana Cu/Zn-SOD genes showed similar expression patterns, and no significant difference in transcriptional level was observed among the three genes. These results increase our understanding of the molecular mechanism of drought tolerance and can be used to improve vegetation restoration programs of Caragana plants.
Keywords: Caragana — Cu/Zn superoxide dismutase — drought stress — gene expression — molecular marker
DOI: 10.7868/S0015330314050182
INTRODUCTION
Adverse habitats, such as those with low or high temperature, water deficit, and soil salinity significantly influence plant growth and crop productivity [1]. Under stress conditions, plant cells can be induced to produce reactive oxygen species (ROS), and overproduction of ROS results in oxygenation of plant membrane lipids and accumulation of malondialdehyde (MDA), which is the cytotoxic end product of membrane lipid peroxidation [2—4]. Through evolution, plants have developed complex molecular mechanisms to survive harsh environments. When under drought stress conditions, plant cells actively accumulate certain types of solutes, such as free proline and soluble sugar, to improve their drought resistance by lowering water poten-
1 This text was submitted by the authors in English.
Abbreviations'. CTAB — cetyltrimethylammonium bromide; Cu/Zn-SOD — copper/zinc superoxide dismutase; ORF — open reading frame; RACE — rapid amplification of cDNA ends; UTR — untranslated region.
Corresponding author. Xiao-Fei Lin. College of Life Sciences, Inner Mongolia University, 235 Daxuexi Road, Hohhot 010021, China; fax. +86-0471-4992435, e-mail. linxiaofei04@hotmail.com
tial and maintaining turgor pressure [5—7]. Plant cells can also increase the expression of antioxidant enzymes to degrade ROS, such as superoxide dismutase (SOD), peroxidase (POD), and low-molecular antioxidants like glutathione (GSH) [8].
SOD is an important antioxidant enzyme that protects organisms against oxidative damage by degrading ROS. The eukaryotic form of Cu/Zn-SOD is present in the cytoplasm and Mn-SOD in the mitochondria. In prokaryotic cells, Cu/Zn-SOD, Mn-SOD, Fe-SOD, and Ni-SOD have been detected [9]. Subcellular frac-tionation studies of plants showed that general plants contain mitochondrial matrix-localized Mn-SOD, cytosolic Cu/Zn-SOD, Fe-SOD, and/or chloroplas-tic Cu/Zn-SOD and Fe-SOD. Seven SODs, two cytosolic Cu/Zn-SODs, one chloroplastic Cu/Zn-SOD, two chloroplastic Fe-SODs, one cytosolic Fe-SOD, and one mitochondrial matrix-localized Mn-SOD, have been indentified in Arabidopsis thaliana [10, 11]. Additionally, eight SODs, two cytosolic Cu/Zn-SODs, two chloroplastic Cu/Zn-SODs, two chloro-plastic Fe-SODs, one cytosolic Fe-SOD, and one mitochondrial matrix-localized Mn-SOD, have been identified in the moss (Physcomitrella patens) [11, 12].
All enzymes appear to be nucleus-encoded, after which they are transported to their organelle locations directed by NH2-terminal targeting sequences [8].
SOD gene regulation is very sensitive to environmental stress (presumably as a consequence of increased oxygen radical formation), such as cold, drought, and salinity [13—17]. Drought stress can induce changes in lipid peroxidation and SOD activities. Drought resistance was found to correlate with Cu/Zn-SOD activity in maize [15]. The drought-tolerant Tortula ruralis showed the lower levels of lipid peroxidation with increased levels of Cu/Zn-SOD enzyme activity compared to the drought-sensitive Cra-toneuron filicinum [18]. In tomato, cytosolic Cu/Zn-SOD was induced strongly by drought, but chloroplastic Cu/Zn-SOD remained largely unaffected (R. Perl-Treves and E. Galun, personal communication) [16]. On the other hand, genetic transformation experiments showed that the overexpression of SOD genes enhanced oxidative and environmental stress tolerance of transgenic plants [19—23]. Therefore, it is very important to identify SOD genes from feral plants with strong adaptability and to characterize their regulatory mechanisms.
Caragana microphylla, C. intermedia, and C. kor-shinskii are native desert shrubs that are widely distributed in the desert land of northwest China and have high forage values and high stress tolerance, as well as sand-fixing capacity. In the Loess Plateau region of northwestern China, the natural vegetation has been seriously degraded during 100 years of human overexploitation. Since the middle of the 20th century, many vegetation restoration programs have been implemented in this region [24]. The plant species of Caragana genus, including C. korshinskii, C. microphylla, and C. intermedia (which have extensive root systems and high stress tolerance) have been widely planted to stabilize eroded land and improve vegetation coverage, and showed preferable ecological benefits and high commercial value. Therefore, a comparative study on the drought-resistant mechanisms of various Caraga-na species is important for vegetation restoration programs.
To date, the expression of SOD genes in Caragana has not been reported; however, Cu/Zn-SOD sequences from C. jubata were registered in the GenBank. In this study, we isolated Cu/Zn-SOD genes from C. microphylla, C. intermedia, and C. korshinskii and investigated their expression under drought stress conditions.
MATERIALS AND METHODS
Plant materials. Mature seeds from three Caragana species, C. korshinskii, C. microphylla, and C. intermedia, were provided by the Alashan Forestry Bureau Seed Orchard, Inner Mongolia, and stored in plastic bags at 4°C for 6—12 months until being used for ex-
periments. The seeds were sown on wet filter paper for germination and transplanted into pots with wet quartz sand, then irrigated with 0.5 Hoagland medium under a photoperiod of 12 h at 22°C. Next, 7-week-old plants were used for various stress treatments.
To investigate the expression of Caragana Cu/Zn-SOD, 0.5 Hoagland medium supplemented with various concentrations of PEG (5, 15, and 25%) or 0.1 M ABA was used to irrigate the Caragana plants for various times, and RNA isolated from the treated plants was used to analyze the expression of Caragana SOD genes.
Isolation of Cu/Zn-SOD genes from three Caragana species. Total RNA was extracted from Caragana plants using the cetyltrimethylammonium bromide (CTAB) method [25]. Caragana seedlings (1.0 g) were ground in liquid nitrogen and transferred to 10 mL of 2x CTAB buffer. The mixture was incubated at 65°C for 30 min and then treated with an equal volume of chloroform : isoamyl alcohol (24 : 1, v/v). After cen-trifugation at 3500 g for 20 min, the supernatant was collected and the RNA was precipitated by adding 0.25 volumes of 10 M LiCl and incubating at -20°C for 2 h. The RNA pellet was collected, dissolved in TE (Tris-EDTA) buffer, and then sequentially treated with equal volumes of TE-saturated phenol, phenol : chloroform : isoamyl alcohol (25 : 24 : 1, v/v/v), and chloroform : isoamyl alcohol (24 : 1, v/v). The RNA was precipitated again by adding 0.25 volumes of10 M LiCl at —20° C for 2 h, and then dissolved in TE buffer.
To isolate SOD cDNAs from Caragana plants, degenerate primers were designed based on Cu/Zn-SOD orthologs from C. jubata (ABQ10188.1), Glycine max (NP_001235298.1), Gossypium hirsutum (ABA00453.1), Medicago truncatula (XP_003 626 362.1), Arachis hy-pogaea (ABF51006.1), Bruguiera gymnorhiza (BAB78597.1), and Ipomoea batatas (AFY26880.1). The primers were SOD-deg-F (5'-ATggTNAARgC-NgTNgCNgT-3') and SOD-deg-R (5'-CCDATDAT-NCCRCANgCNAC-3'), which corresponded to the highly conserved amino acid sequences MVKAVAV and VACGIIG. Reverse transcription-polymerase chain reaction (RT-PCR) was performed using a Takara RT-PCR Kit ("Takara Bio", China) and the degenerate primers to isolate SOD cDNA fragments. First-strand cDNA was synthesized from 1 |g of total RNA at 42°C for 60 min with 1 |L of oligo dT-Adap-tor ("Takara Bio"). The PCR reaction was performed with 2.5 |L of 10x Ex Taqbuffer ("Takara Bio"), 2 |L of 2.5 mM dNTPs, 1 U Ex Taq HS DNA polymerase ("Takara Bio"), 1 |L of 50 |M primer mix (SOD-deg-F and SOD-deg-R), and 1 |L ofcDNA in a 25-|L volume with the following conditions: 35 cycles at 94°C for 30 s, 45°C for 30 s, and 72°C for 1 min,
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