научная статья по теме OVEREXPRESSION OF DNWRKY29 IN TOBACCO IMPAIRED PLANT TOLERANCE TO SALT AND DROUGHT STRESSES Биология

Текст научной статьи на тему «OVEREXPRESSION OF DNWRKY29 IN TOBACCO IMPAIRED PLANT TOLERANCE TO SALT AND DROUGHT STRESSES»

ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ

УДК 581.1

OVEREXPRESSION OF DnWRKY29 IN TOBACCO IMPAIRED PLANT TOLERANCE TO SALT AND DROUGHT STRESSES1

© 2015 X. Xu, C. Wang, X. Ma, Y. Pan, Q. Ying, H. Song, H. Wang

College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, China

Received May 22, 2014

One novel transcription factor gene DnWRKY29 was isolated from Dendrobium officinale, and its functions were identified by it overexpressing in tobacco. Compared to the wild type (WT), the DnWRKY29 transgenic tobacco seeds and seedlings showed the higher sensitivity to salt and drought stresses. The seed germination rate, seedling root length, and fresh weight of transgenic tobacco were lower than in WT. The activities of per-oxidase (POD), superoxide dismutase (SOD), and catalase (CAT) in transgenic tobacco were decreased, and the content of malondialdehyde (MDA) in transgenic tobacco was increased. Moreover, the expression of DnWRKY29 was up-regulated in Dendrobium seedlings under salt and drought stresses, which is consistent with the function identified by its overexpression in tobacco, indicating that the up-regulation or overexpression of DnWRKY29 impaired plant tolerance to salt and drought stresses. These results provided a new insight to the low tolerance of Dendrobium seedlings to environmental changes.

Keywords: Dendrobium - stress transcription factor - WRKY

DOI: 10.7868/S0015330315010170

INTRODUCTION

Plants are constantly challenged by various environmental changes, such as drought, salt, extreme temperatures, and oxidative stress. To survive these challenges, they have developed elaborate mechanisms to adapt themselves [1]. WRKY transcription factors are one of the largest families of transcriptional regulators of plant response to biotic and abiotic stresses. They are defined by the presence of one or two WRKY conserved domains of 60 amino acids characterized by the WRKYGQK motif at the N-ter-minus, and a Cys2His2 or Cys2HisCys zinc-binding motif at the C-terminus [2]. Based on the number of WRKY domains present and the type of the zinc-binding motif, WRKY proteins were classified into groups 1, 2a to 2e, and 3 [2]. Up to date, a large number of WRKY genes have been identified in plants [2-6]. They are involved in various stress responses, developmental and physiological processes, such as anthocya-nin biosynthesis, senescence, trichome development, starch biosynthesis, and hormone responses [1, 7-13].

1 This text was submitted by the authors in English.

Abbreviations: CAT - catalase; POD - peroxidase; SOD - superoxide dismutase; WT - wild type.

Corresponding authors: Xiangbin Xu and Huizhong Wang. College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, China; fax: +86-571-2886-5330; e-mails: xbxuibcas@126.com, whz62@163.com

Dendrobium is the second largest genus in the family Orchidaceae, which comprises over 1100 species, and 74 species have been identified in China. Some members of these species, such as Dendrobium candi-dum, D. nobile, and D. chrysanthum, containing various bioactive constituents, which makes them highly valued in Chinese traditional herbal medicine. Since their important role in disease treatment and tonic effect, during the last few decades, wild Dendrobium suffered excessive collection for growing medicinal demands, and fallen into serious danger of extinction due to the limited natural resource in China. In recent years, Dendrobium seedlings have been obtained via tissue culture technology. However, the seedlings often grow slowly and show a low survival rate when transferred from in vitro conditions to a greenhouse or field environment, which seriously affects the economic benefits. The major reason is their low tolerance to environmental changes. Hence, studying the mechanism of their low tolerance to environmental changes and searching for the stress-related genes of Dendrobium is important for improving their survival rate and growth.

In the present study, one novel DnWRKY29 was isolated from D. officinale; its function in plant response to abiotic stresses was analyzed by overexpressing it in tobacco.

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MATERIALS AND METHODS

WRKY gene cloning. Total RNA was extracted from Dendrobium officinale roots. First-strand cDNA was synthesized with first-strand cDNA synthesis kit ("TianGen", China) and used for PCR. Degenerate PCR primers were designed based on the WRKY conserved coding region. Primers sequences were: 5'-ATGGC(C/T)GT(C/T)GATCT(A/C)ATCGG(A/ C)TAC-3' (forward), 5'-TCAGATC(A/T)CG-CG(G/T)TG(A/G)G(G/T)(A/G)TC(A/G)A(A/G)-GACGA-3' (reverse). PCR conditions were: 94°C for 3 min, 30 cycles of 94°C for 30 s, 60°C for 30 s, 72°C for 90 s; and finally 72°C for 10 min. The resulting PCR product was cloned and sequenced. The nucleotide sequence of the full-length cDNA was analyzed by using blast search of the GenBank Database at NCBI.

PCR analyses of DnWRKY29 in Dendrobium seedlings under salt and drought stresses. To understand the function of the DnWRKY29 in plant response to salt and drought stresses, the D. officinale seedlings were submerged into 0.5-strength MS agar plates containing distilled water, 200 mM NaCl or 200 mM mannitol, and cultured in a greenhouse (25°C, 16 h of light and 8 h of darkness) for 24 and 48 h. The expression of DnWRKY29in D. officinale seedlings was analyzed by real-time PCR. PCR amplifications were performed three times for each RNA sample using primers of DnWRKY29 P1 and P2. To verify the quality of cDNAs from different tissue samples, actin cDNA was amplified as an internal control, using primer pairs P5 (5'-CAGGCATGTGTTGGAT-TCT3') and P6(5'-ACAACAGCAGAACGGGAAT3') based on actin mRNA sequence from our previous cDNA library. Real-time PCR amplification conditions consisted of an initial denaturation step at 94°C for 3 min, followed by 40 cycles of 20 s at 94°C, 20 s at 55°C, and 23 s at 72°C, with a fluorescence detection at 80°C for 2 s.

Plasmid construction and tobacco transformation.

The DnWRKY29 was inserted into pEASY-T1 vector ("TianGen"), sequenced, and then moved into the SacI and BamHI sites of pSN1301 vector to create the plant expression vector pSN1301-DnWRKY29. Then the plant expression vector pSN1301-DnWRKY29 was introduced into Agrobacterium tumefaciens GV3101 by electroporation. Nicotiana tabacum cv. Huangmiaoyu was used for gene transformation. T1 seeds were collected, dried at 25°C, and sown on sterile medium containing 80 ^g/mL hygromycin to select the transformants. Survived T1 plants were transferred to soil to obtain seeds (T2). T3 seeds were obtained by the same way.

PCR analysis of transgenic plants. To confirm transgenic tobacco, genomic DNA was extracted from the screened tobacco, and PCR analysis was conducted to test transgenic insertion. Two primers, GUS-5 (5' - GCATGTTACGTCCTGTAGAAACCC - 3') and GUS-3 (5'-CAAAGCCAGTAAAGTAGAACGGT-3'), were designed to target the regions of the GUS report

gene. Using tobacco genomic DNA as a template, PCR was performed with a predenaturing conditions of 94°C for 3 min, 30 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 80 s; and finally 72°C for 10 min.

The expression of DnWRKY29 in transgenic tobacco was analyzed by real-time PCR. Total plant RNA was extracted from transgenic tobacco using Trizol reagent ("TianGen"). PCR amplification was performed three times for each RNA sample using primers of DnWRKY29 P1 (5'-TCAACGACAACGG-GAACG-3') and P2 (5'-ATGGAGGGCGG-AGGAAGGA-3'). To verify the quality of cDNAs from different tissue samples, actin cDNA was amplified as an internal control using primer pairs P3 (5'-ATGGCGGATGGGGAGGACATT-3') and P4 (5'-TTAGAAGCATTTGCGGTGGAC-3') based on N. tabacum actin mRNA (GenBank accession no. AB158612.1). Real-time PCR amplification conditions consisted of an initial denaturation step at 94°C for 3 min, followed by 40 cycles of 20 s at 94°C, 20 s at 55°C, and 23 s at 72°C, with a fluorescence detection at 80°C for 2 s.

Stress tolerance assay of transgenic plants. To evaluate the functions of the DnWRKY29 in plant response to salt and drought stresses, the aseptic tobacco seeds of WT and three transgenic lines were submerged into 0.5-strength MS agar plates containing distilled water, 200 mM NaCl or 200 mM mannitol, and cultured in a greenhouse (25°C, 16 h of light and 8 h of darkness). The germination rates, root lengths, and fresh weights were measured.

Assay of antioxidant enzyme activities and MDA content. To test the antioxidant defense response of DnWRKY29 transgenic plants to abiotic stresses, three-week T1 DnWRKY29 transgenic seedlings were placed on Hoagland medium supplemented with NaCl (200 mM) or mannitol (200 mM) for 24 h. The transgenic seedlings treated with distilled water were used as control. Seedling tissue (1 g) was collected and homogenized in 5 mL of ice-cold extraction buffer and 0.1 g of polyvinyl polypyrrolidone. For catalase (CAT) analysis, 50 mM sodium phosphate (pH 7.0) was used as an extraction buffer. For superoxide dis-mutase (SOD), peroxidase (POD), and MDA content measurements, 100 mM sodium phosphate buffer (pH 6.4) was used as extraction buffer. The activities of CAT, POD, SOD, and the content of MDA were measured according to the methods of Song et al. [14].

RESULTS

Dnwrky29 gene cloning

By using the method of rapid-amplification of cDNA ends (RACE), one gene was cloned from D. officinale. By blast against NCBI, it was designated as DnWRKY29 (GenBank accession no. KF746197). Its full-length cDNA contained 930 bp and encoded 309 amino acids. The deduced DnWRKY29 possessed

one WRKY domain that contained the highly conserved amino acid sequence WRKYGQK and one Cys2His2 zinc finger motif; it belongs to group 2 WRKY transcription factors.

DnWRKY29expression in Dendrobium seedlings under salt and drought stresses

The expression of DnWRKY29 in D. officinale seedlings under salt and drought stresses was analyzed by real-time PCR. Results showed that the expression

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