ФИЗИОЛОГИЯ РАСТЕНИЙ, 2014, том 61, № 5, с. 705-712
^ ЭКСПЕРИМЕНТАЛЬНЫЕ
СТАТЬИ
УДК 581.1
CLONING AND CHARACTERIZATION OF PATHOGENESIS-RELATED PROTEIN 4 GENE FROM Panax ginseng1
© 2014 Y. J. Kim2, H. J. Lee2, M. G. Jang, W. S. Kwon, S. Y. Kim, D. C. Yang
Department of Oriental Medicinal Materials and Processing, College of Life Science, Kyung Hee University, Korea
Received October 11, 2013
The family of pathogenesis-related protein 4 (PR4) is a group of proteins with a Barwin domain in C-termi-nus and generally thought to be involved in plant defense responses. In the present study, PR4 (designated as PgPR4) cDNA was isolated from the leaf of Panax ginseng C.A. Meyer and characterized. The ORF is 513 bp with a deduced amino acid sequence of 170 residues. A GenBank BlastX search revealed that the deduced amino acid of PgPR4 shares the highest sequence similarity to PR4 of Sambucus nigra (72% identity). Sequence and structural analysis indicated that PgPR4 belongs to class II of PR4 proteins. This is the first report on the isolation of PR4 gene from the P. ginseng genome. The high-level expression of PgPR4 was observed in the root as revealed by quantitative real-time PCR. The temporal expression analysis demonstrated that PgPR4 expression could be up-regulated by pathogen infection, salt, wounding, and hormone stresses. These results suggest that PgPR4 could play a role in the molecular defense response of ginseng to abiotic stress and pathogen attack.
Keywords: Panax ginseng — abiotic stress — biotic stress — pathogenesis-related protein 4 — gene expression
DOI: 10.7868/S0015330314050108
INTRODUCTION
Higher plants have developed various defense mechanisms against biotic and abiotic stresses, such as pathogen invasions, wounding, exposure to heavy metal, salinity, cold, and ultraviolet rays (UV). These defense mechanisms include the synthesis of patho-genesis-related (PR) proteins. When the pathogens attack, the generation of reactive oxygen species (ROS) strengthens plant cell walls, PR proteins are induced, phytoalexins are accumulated, and antimicrobial compounds are synthesized [1]. PR proteins are known to function in higher plants against abiotic and biotic stresses, especially against pathogen infection. PRs are accumulated after the attacks by virus, bacteria, fungi, nematodes, insects, and herbivores as well as after wounding and certain abiotic stress conditions [2]. PR was first observed in tobacco plants infected with tobacco mosaic virus [3]. Since the discovery of PRs in 1970, PRs were identified in many plants species; currently, 17 PR families have been grouped
1 This text was submitted by the authors in English.
2 These authors contributed equally to this work.
Abbreviations'. Ct — threshold cycle; JA — jasmonic acid; PR — pathogenesis related; SA — salicylic acid.
Corresponding author. Deok-Chun Yang. Department of Oriental Medicinal Materials and Processing, College of Life Science, Kyung Hee University, Suwon 449-701, Korea; fax. +82-31-2022687; e-mail. dcyang@khu.ac.kr
based on amino acid sequences, serological relationship and/or enzymatic or biological activity [2]. The specific functions of PRs are not fully understood, although several proteins are postulated to play a role in preventing pathogen invasion.
Among the 17 groups of PR proteins, pathogene-sis-related protein 4 (PR4) family proteins contain a common barwin domain in C-terminus, derived from a basic barley seed protein barwin, which contains six cysteine residues that can form three disulfide bridges and has the ability to bind saccharides [4]. PR4 proteins have been regarded as endochitinases because one of them, tobacco CBP20 [5], reported to manifest weak chitinase activity [6]. PR4 proteins can be divided into two classes according to the presence of the cysteine-rich domain in N-terminus [7]. Class I PR4 proteins (also known as hevein-like proteins) contain a conserved N-terminal cysteine-rich chitin-binding domain (or hevein domain), which corresponds to an antifungal protein from rubber tree latex (Hevea bra-siliensis) [8]. They possess binding ability to chitin and thereby have strong antifungal activities [9]. However, class II PR4 proteins, which lack the chitin-binding domain, are less extensively studied and their functions are divergent. They are reported to be involved in defense responses against various biotic or abiotic stresses [10]. At present, the role of PR4 protein in defense of ginseng remains to be elucidated.
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In this study, we isolated cDNA (PgPR4), for Panax ginseng PR4 protein, which shared significant sequence similarity with PR4 from other plants. Korean ginseng (Panax ginseng C.A. Meyer) is cultivated for its highly valued root used for medicinal purposes. The production of ginseng roots required 4-6-year cultivation period; thus it is sensitive to environmental stresses as well as diseases caused by both foliar and soil-born root-infecting fungi [11, 12]. To effectively manage ginseng diseases and other disorders, it is valuable to study and identify functional genes related to defense mechanism in ginseng. To understand the defense response of ginseng to stress, we have isolated PR genes from ginseng. In this study, we report on the cloning of PR4 gene from P. ginseng and provide the analysis on the expression profile of this gene in the defense responses to abiotic and biotic stresses.
MATERIALS AND METHODS
RNA purification and construction of a cDNA library. Total RNA was isolated from ginseng callus (provided by Ginseng Bank) using the aqueous phenol extraction procedure as described by Morris et al. [13]. Poly(A) + RNA was isolated by using the oligo (dT) cellulose column using the Poly(A) Quick mRNA isolation kit ("Stratagene", United States). A cDNA synthesis kit was used to construct library according to the manufacturer's instruction manual ("Clontech", United States). Size-selected cDNA was ligated into ^TriplEx2 vector and was packaged in vitro using Gi-gapack III Gold Packaging Extract kits ("Stratagene").
Nucleotide sequencing and sequence analysis.
pTriplEx phagemids were excised from the ^pTriplEx2 and used as templates for sequence analysis. The 5'-ends of cDNA inserts were sequenced by an automatic DNA sequencer (ABI prism 3700 DNA sequencer, "Perkin-Elmer", United States). Homologous sequences ofPR4 EST were searched against the GenBank databases using a BLASTX algorithm. A pTriplEx phagemid for PR4 cDNA was excised from the ^pTriplEx2 and used as templates for sequence analysis. Nucleotide and amino acid sequence analyses were performed using DNASIS program ("Hitachi", Japan).
The deduced amino acid sequences were searched for homologous proteins in the databases using BLAST network services at the NCBI. We used ClustalX with default gap penalties to perform multiple alignment of PR4 isolated in ginseng and previously registered in other species. Based on this alignment, a phylogenetic tree was constructed according to the neighbor-joining method using the MEGA4 programs. The protein properties were estimated using ProtParam [14]. Identification of conserved motifs within PR4 was accomplished with MEME [15]. A
three-dimensional model was prepared using PR4 as a template on a SWISS-MODEL WORKSPACE in automated mode [16]. The generated 3D structure was visualized using the UCSF Chimera package.
Plant, environmental stress, and pathogen. Panax ginseng C.A. Meyer cv. Hwang-Sook seeds (provided by Ginseng Bank) were used, and cultivated three-week-old plantlets were used for the treatments and RNA extraction, as described previously [17]. For chemical stress treatments, the plantlets were placed for various periods on MS medium containing indicated concentrations of chemicals: 100 mM NaCl, 10 mM H2O2, 0.1 mM abscisic acid (ABA), 0.2 mM jasmonic acid (JA), and 0.2 mM salicylic acid (SA). Chilling stress was applied by exposing the plantlets to 4°C. For mechanical wounding stress, healthy leaves and stems of plantlets were wounded with a sterile scalpel. In all cases, stress treatments were carried out on MS media and 10 plantlets were treated with each stress for 1, 4, 8, 24, 48, or 72 h.
The fungal strains, Colletotrichum gloeosporoides (KACC 40003) and Rhizoctonia solani (KACC 40101), were obtained from Korean Agricultural Collection Center, South Korea. The isolates of fungi were grown for three days at 25°C on potato dextrose agar to obtain mycelium for the inoculation of ginseng seedlings. For the infection experiments, mycelial plug excised from the actively growing margin of a colony were suspended in sterile water and sprayed. After inoculation, plants were kept at 100% humidity to attain moisture condition. Plants were harvested at 0, 6, 24, 48, and 72 h after infection. Control plants were held in a growth room at 25°C under a 16-h photope-riod. The stressed plant material from all completed treatments were immediately frozen in liquid nitrogen and stored at —70°C until required.
Real-time quantitative RT-PCR. Total RNA was extracted from seedlings of P. ginseng using RNeasy mini kit ("Qiagen", United States). For RT-PCR, 200 ng of total RNA was used as a template for reverse transcription using oligo(dT)15 primer (0.2 mM) and AMV Reverse Transcriptase (10 U/|L) ("iNtRON Biotechnology", South Korea) according to the manufacturer's instructions. Real-time quantitative PCR was performed using 100 ng of cDNA in a 10-|L reaction volume using SYBR® Green SensimixPlus Master Mix ("Quantace", England). Specific primers for PgPR4, 5'-ATGGACAGCCTTCTGTGGTC-3' and 5'-CTTGAGCATTACCCCTTCCA-3' were used for perform real-time PCR. The thermal cycler conditions recommended by the manufacturer were used as follows: 10 min at 95°C, followed by 40 cycles of 95°C for 10 s, 58°C for 10 s, and 72°C for 20 s. The fluorescent product was detected at the last step of each cycle. Amplification, detection, and data analysis were carried out with a Rotor-Gene 6000
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