KR101876775B1 - Drought and Osmotic Stress Registant Gene and Transformant Using the Same - Google Patents

Abstract

The present invention relates to a dry and osmotic stress resistance gene having a predetermined homology with the nucleotide sequence of SEQ ID NO: 1 derived from Kimchi and a transformant having a dry and osmotic stress resistance using the same.
The gene according to the present invention is a gene induced by dry or osmotic stress, and is expressed in a bird or a green plant, thereby increasing the resistance to dryness and osmotic stress of a bird or a plant.

Description

{Drought and Osmotic Stress Registant Gene and Transformant Using the Same}

The present invention relates to a gene having resistance to dry and osmotic stress derived from Kimchi and a transformant having dry and osmotic stress resistance using the same.

Kim ( Pyropia tenera (Bangiales, Rhodophyta)) is one of commercially important marine red algae cultivated in Korea, Japan and China. P. tenera is exposed to repeated and extreme stressful environmental conditions such as 80 to 95% water loss during blast (Blouin et al. 2011) and water replenishment during tide. Therefore, P. tenera can be expected to have various strategies and mechanisms for survival under dehydration stress conditions.

So far, studies on dry and osmotic stress tolerance genes have been conducted mainly on land plants. Phylogenetic studies show that red algae have been systematically branched into green plants very long ago. Therefore, it is presumed that there is a flora-specific mechanism in addition to a mechanism common to the dry and osmotic stress mechanisms appearing in the land plants.

However, little is known about the mechanism of dehydration resistance of red algae in the intertidal zone at the molecular level, and recent work on this has focused on the role of reactive oxygen species (Burritt et al., 2002; Contreras-Porcia et al 2011; Kumar et al., 2011). ROS is known to play a multifaceted role in signal and stress responses because it can produce oxidized polyunsaturated fatty acids that protect against oxidative stress (Foyer and Noctor 2005). Carbohydrates and polysaccharides also participate in the protection of cell membranes and proteins in osmotic and water-loss stress environments (Ghasempour et al. 1998; Tamaru et al. 2005). In particular, photosynthetic carbohydrates, floridosides, accumulate to protect cells in dehydrated stress environments in Pyropia (Reed et al., 1980; Qian et al. However, little is known about the stress signaling pathways and downstream genes involved in dehydration stress in red algae.

Recently, there have been many studies on the production of biodiesel feeds using microalgae, the fixation of atmospheric carbon dioxide by mass culture of marine microalgae, and the development of plant food for fish culture aquaculture. Whether the selected microalgae have broad adaptability to the concentration of the media in these studies is an important factor in the determination of industrial practicality.

Dry stress in terrestrial crops is known to be the most important abiotic environmental stress limiting temperature stress and crop growth and production. For example, these abiotic environmental stresses have been found to result in a loss of more than half of possible crop production.

Blouin NA, Brodie JA, Grossman AC, Xu P, Brawley SH (2011) Porphyra: a marine crop shaped by stress. Trend in Plant Sci 16: 29-37 Burritt DJ, Larkindale J, Hurd CL (2002) Antioxidant metabolism in the intertidal red seaweed Stictosiphonia arbuscula following desiccation. Planta 215: 829-838 Contreras-Porcia L, Thomas D, Flores V, Correa JA (2011) Tolerance to oxidative stress induced by desiccation in Porphyra columbina (Bangiales, Rhodophyta). J Exp Bot 62: 1815-1829 Kumar M, Gupta V, Trivedi N, Kumari P, Bijo AJ, Reddy CRK, Jha B (2011) Desiccation induced oxidative stress and its biochemical responses in intertidal red alga Gracilaria corticata (Gracilariales, Rhodophyta). Environ Exp Bot 72: 194-201 Foyer CH, Noctor G (2005) Oxidant and antioxidant signaling in plants: a reevaluation of the concept of oxidative stress in a physiological context. Plant Cell Environ 28: 1056-1071 Ghasempour HR, Gaff DF, Williams RPW, Gianello RD (1998) Contents of sugars in leaves of drying desiccation tolerant flowering plants, especially grasses. Plant Growth Regul 24: 185-191 Tamaru Y, Takani Y, Yoshida T, Sakamoto T (2005) Crucial role of extracellular polysaccharides in desiccation and freezing tolerance in the terrestrial cyanobacterium Nostoc commune. Appl Environ Microbiol 71: 7327-7333 Reed RH, Collins JC, Russell G (1980) The effects of salinity on galactosyl-glycerol content and concentration of the marine red alga Porphyra purpurea (Roth). C. Ag. J Exp Bot 31: 1539-1554 Qian F, Luo Q, Yang R, Zhu Z, Chen H, Yan X (2015) The littoral red alga Pyropia haitanensis uses rapid accumulation of floridoside as the desiccation acclimation strategy. J Appl Phycol 27: 621-632 Hwang MS, Kim SM, Ha DS, Baek JM, Kim HS, Choi HG (2005) DNA sequences and identification of Porphyra cultivated by natural seeding on the southwest coast of Korea. Algae 20: 183-196 McLachlan J (1973) Growth media-marine. In: Stein JR (ed) Handbook of Phycological methods. Cambridge Univ Press, New York, pp 25-51 Im S, Choi S, Hwang MS, Park EJ, Jeong WJ, Choi DW (2015) De novo assembly of transcriptome from the gametophyte of the marine red algae Pyropia seriata and identification of abiotic stress response genes. J Applied Phycology 27: 1343-1353

The present invention aims to find a resistance gene against environmental stresses such as osmotic stress, dehydration stress and the like on a plant such as a microalgae or a terrestrial crop and apply it to microalgae or land crops.

In order to achieve the above object, the present invention is characterized in that it is a dry and osmotic stress resistance gene comprising the nucleotide sequence of SEQ ID NO: 1. Further, the present invention may be a sequence having a homology of at least 80%, preferably at least 90%, more preferably at least 95% with SEQ ID NO: 1.

The gene can be isolated from the genus Pyropia .

The present inventors have identified a number of genes corresponding to dehydration stress by generating transcript sequences from the thallus of P. tenera under the control and dehydration conditions, and identified the genes corresponding to the sequence 1 (Fig. 1) as PtDRG2 Respectively.

The present invention also relates to a dry and osmotic stress-resistant transformation vector comprising the gene and a dry and osmotic stress-resistant transformant into which the transformation vector is introduced.

The transformation vector may further include a selection marker capable of confirming the expression of the gene of the present invention or selecting a transformant. Examples of the selection marker include genes showing resistance to antibiotics such as kanamycin, hygromycin, gentamycin and bleomycin or genes expressing GUS (β-glucuronidase), CAT (chloramphenicol acetyltransferase), luciferase or GFP ), And the like, but is not limited thereto.

In the present invention, the transformed vector may be a vector expressed in an alga such as, for example, a pCr112 vector, and the transformant may be a bird. In the following example, the gene was cloned into pCr112 vector and introduced into Clamidomonas to obtain a dry and osmotic stress resistant transformant.

The present invention also relates to a dry and osmotic stress resistant protein encoded by the gene and a method of using the protein for the purpose of enhancing dryness or osmotic stress resistance of birds or plants.

As described above, the gene according to the present invention isolated from laver is a gene induced by dry or osmotic stress, and can be expressed in algae or green plants, thereby enhancing the drying and osmotic stress resistance of algae or plants.

1 and 2 are respectively the nucleotide sequence of the gene PtDRG2 according to the present invention and the amino acid sequence of the protein encoded thereby .
Fig. 3 is an amino acid sequence comparison between a protein encoded by the gene PtDRG2 according to the present invention and a similar protein of Pyropia species. Fig.
FIGS. 4 and 5 are graphs and photographs showing that PtDRG2 according to the present invention increases expression in dehydration stress and encodes chloroplast proteins. FIG.
6 is a conceptual diagram of a region in which PtDRG2 is introduced into a pCr112 vector in an embodiment of the present invention.
FIG. 7 is a photograph of the electrophoresis result of the PCR product showing that PtDRG2 according to the present invention was introduced into Clamidomonas cells.
FIG. 8 is a photograph showing the result of an experiment in which a plastid transformed with PtDRG2 according to the present invention shows resistance to a salt or mannitol.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail with reference to the accompanying drawings and embodiments. However, the drawings and the embodiments are only illustrative of the contents and scope of the technical idea of the present invention, and the technical scope of the present invention is not limited or changed. It will be apparent to those skilled in the art that various changes and modifications can be made within the scope of the technical idea of the present invention based on these examples.

The present invention was obtained as a result of identifying and analyzing the gene PtDRG2 encoding a novel dehydration reaction protein from P. tenera .

In order to identify new genes involved in dehydration resistance in P. tenera , a large number of transcript sequences were obtained from the control and dry stressed thalli, and compared with the control and dry stress-treated transcriptome (DEGs) genes were selected and named dehydration-reacting genes ( PtDRGs ). Of the selected PtDRGs , PtDRG2 (FIG. 1) encoding a polypeptide of 159 amino acids (SEQ ID NO: 2, FIG. 2) was cloned and its physiological function was studied. Although PtDRG2 did not show sequence homology with proteins known in public databases, its homologues were detected in a local transcript database of P. yezoensis and P. seriata (Fig. 3). These results suggest that PtDRG2 may be pyropia - specific or its homologues in green plants may be low. PtDRG2 was found to encode a new chloroplast protein and increased expression in dehydration stress (see Figures 4 and 5). Therefore, PtDRG2 is thought to be involved in resistance mechanism against abiotic stress such as dehydration.

To confirm the physiological function of PtDRG2 , this was introduced into Clamidomonas and the tolerance to abiotic stress of the transfected cells was analyzed. The PtDRG2 gene transgenic clomidomonas grew better than control cells with empty vectors in agar medium containing high salt or mannitol (Fig. 8). These results demonstrate that the PtDRG2 gene encodes a small new chloroplast protein and contributes to the osmotic stress tolerance mechanism by the mannitol and salt stress of the clamidomonas.

[Example]

(1) Degranulation and dehydration reaction

Pyropia tenera (Kjellman, LB) used in this study was obtained from the seaweed research center of the National Fisheries Research and Development Institute (Hwang et al. 2005). Dehydration stress was induced by the method of Lim et al. (2015), and the thalli were frozen in liquid nitrogen and stored at -80 ° C until RNA extraction.

Generation of transcripts from the control and dehydrated thalli were determined using the GS-FLX 454 platform in G & C Bio (Daejeon, Korea). Briefly, the cDNA library for GS-FLX 454 sequencing was generated using a cDNA synthesis kit (Roche, Switzerland). About 1 μg of poly (A) ⁺ RNA was used for cDNA synthesis. Adapter-linked cDNA was coupled to beads, amplified by emulsion PCR, and generated a nucleotide sequence using the GS-FLX 454 platform. Very short (<50 BP) or low-confidence reads among the generated transcript reads were removed and De novo assembled using the Genomics Workbench 7.5.1 (CLC bio, Germany) program.

The 13,170 cones obtained from de novo assemblies were judged to be dehydration reaction DEGs based on RPKM values, showing contiguous changes of more than 2 times in dehydrated stressed fronds compared to the control. A total of 1,160 conti was judged to be DEG reacting to dehydration.

Sixteen dehydrated DEGs selected from control and dehydrated stressed livers were selected and their expression levels were investigated experimentally using qRT-PCR. A comparison of the relative expression levels of selected DEGs showed that expression of all genes increased more than twice after dehydration stress. Thus, 87.5% of the genes selected by sequence reads were confirmed by qRT-PCR.

(2) PtDRG2  Cloning of genes

As a result of qRT-PCR, the contigs containing no open reading frame sequence with known proteins in the identified DEGs which were found to be increased by dehydration stress, and contigs containing the open reading frame for the polypeptide were selected as dehydration reaction genes and named as PtDRGs . The open reading frame was searched with Sequencher software (GeneCode Corporation, Ann Arbor, Mich., USA) and the cDNA for the entire ORF was PCR amplified (Figure 1). The PCR product was cloned into pGEM T-easy vector (Promega, USA) and sequenced. The estimated molecular weights and pI values of the selected genes were estimated using ProtParam (http://web.expasy.org/compute_pi/).

In the present invention, it was confirmed that among the selected PtDRGs genes, the PtDRG2 cDNA encodes a polypeptide having a molecular weight of 17.54 kDa consisting of 159 amino acids and a pI of 5.20 (SEQ ID NO: 2) (FIG. 2). Alanine (1.3 mol%) was the most abundant in the PtDRG2 polypeptide. In addition, the Pyropia species P. yezoensis and P. seriata cultured in Asia PtDRG2 ortholog (Fig. 3). PtDRG2 was identical to PyDRG2 of P. yezoensis except for one amino acid. However, it showed 88.4% amino acid sequence identity with PsDRG2 (Fig. 3) of P. seriata (Fig. 3).

(3) PtDRG2 Gene expression analysis

To confirm the gene expression pattern of PtDRG2 , total RNA was isolated from the control and various stress-treated tissues and used for qRT-PCR. Dehydration stress was treated for 4 hours in the same manner as described above. For treatment with mannitol and H ₂ O ₂ , gametophytes were cultured in medium containing 0.5 M mannitol or 20 mM H ₂ O ₂ for 8 hours. Heat and freezing stress were induced at 20 &lt; 0 &gt; C and 4 &lt; 0 &gt; C for 4 hours, as described by Lim et al. (2015). After DNase I treatment, 1.6 μg of total RNA was reverse transcribed with single strand cDNA in a 40 μl reaction volume according to the manufacturer's instructions using AmfiRivert reverse transcriptase (Gendepot, USA). Sequence specific primer sets from selected contigs were designed using the Primer 3 program. Actin genes (5'-TCCTGGGCAATGAGATGAGC-3 'and 5'-AATCCACACCGAGTACTGCC-3') were used as internal controls. QRT-PCR was performed according to the description of Choi et al. (2013). As a result of qRT-PCR, the expression of PtDRG2 gene was increased about 20 times in the thirst -treated thallus, and the expression was also increased in mannitol and hydrogen peroxide treated thrips. However, it did not respond to high temperature or icing stress (Fig. 4). These results show that the resistance mechanism of osmotic stress induced by the PtDRG2 gene induced by drying or mannitol is involved.

(4) PtDRG2 Of cells

Prediction of the intracellular location of PtDRG2 suggests that PtDRG2 may be located in a cell organ such as chloroplast. (SEQ ID NO: 3: 5'-TCTAGATGGTCAAGTTTTCCAAGACG-3 ') and reverse direction (SEQ ID NO: 4: 5'-GGATCCACAACAAGTTTTTTGTCACAAA-3') primers containing XbaI and BamHI recognition sites to determine the intracellular position of PtDRG2 protein To amplify the PtDRG2 coding region and to clone Pt DRG2 between the 35S promoter and the green fluorescent protein (GFP) gene of the plant expression vector 326-GFP. The chimeric PtDRG2 - GFP expression construct was injected into Nicotiana bethamiana protoplast, followed by fluorescence microscopy to confirm GFP expression and intracellular location (change).

Fluorescence of the PtDRG2-GFP fusion protein was mainly observed in chloroplasts (Fig. 5). These results indicate that PtDRG2 encodes small chloroplast proteins.

(5) PtDRG2 Production of transformed Clamidomonas

The Chlamydomonas reinhardtii strain Mut11 was used to analyze the physiological function of PtDRG2. Chlamydomonas cells 25 ℃, 14:10 (name: f) cooling the fluorescent photoperiod ^- were incubated with shaking at ^{(50 mmol photon m -2 s 1} ) under a Tris-acetate-phosphate (TAP) medium with 100 rpm. The PtDRG2 gene ORF was amplified using the forward (SEQ ID NO: 5: 5'-CATATGGTCAAGTTTTCCAAGACG-3 ') and reverse direction (SEQ ID NO: 6: 5'-GATATCTCACAACAAGTTTTTTTCAC-3') primers containing the NdeI and EcoRV recognition sites and pCr112 vector was cloned into the Nde I and Eco RV sites under the PsaD promoter (Figure 6). The pCr112- PtDRG2 plasmid was introduced into C. reinhardtii . Clamidomonas transformants were selected after culturing for 7-14 days in TAP agar medium containing 10 mg mL ^-1 hygromycin. The transformed clamidomonas cell line was screened by hygromycin resistance. To confirm the introduction of PtDRG2 in the transformation line, genomic DNA was extracted from a hygromycin resistant transfection line and PCR was performed with a PtDRG2 specific primer (Figure 7). The PtDRG2 gene was detected in all antibiotic-resistant clamidomonas, but no amplification band was observed in the control cells transfected with the empty vector (Hyg) (Fig. 7). Finally, 4 transformed clamidomonas cell lines were selected and used for additional abiotic stress tolerance assays.

(6) Identification of abiotic stress tolerance of transformed clamidomonas

Clamidomonas cells carrying the PtDRG2 gene were cultured at a concentration of about 2-4 × 10 ⁶ cells / ml and diluted 10 ¹ -10 ³ times in fresh TAP medium. The diluted cells are inoculated 10 ㎖ and 25 ℃ on an agar plate medium, 14:10 (n: f) cooling the fluorescent ^{photoperiod-were} cultured in chamber under ^{(50 mmol photon m -2 s 1} ). For osmotic stress treatment, the cells were inoculated into TAP medium containing 200 mM mannitol. For salt treatment, the cells were inoculated into TAP medium containing 80 mM NaCl. The C. reinhardtii strain Mut11 with empty pCr112 vector (Hyg) was used as a control.

The transformed clamidomonas cells showed a higher growth rate in the salt or mannitol-containing medium than in the control cells (Fig. 8). These results indicate that PtDRG2 is involved in the resistance mechanism against osmotic stress induced by high salt and mannitol in transformed Clamidomonas cells. Studies show that PtDRG2 is a novel gene that is not similar to any of the genes reported so far and is involved in osmotic and salt stress tolerance.

<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Drought and Osmotic Stress Registant Gene and Transformant Using          the Same <130> P1016-616 <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 802 <212> DNA <213> Pyropia tenera <400> 1 ctgcccgcct cctgcgtcgt catgcccgtc tcgtggcgtt gtacactgcc gtcatcgtcc 60 caactgcatc cgggatttcc ctgttctttc tgctctgcct gccagcgtcg cactgccgaa 120 ggtgtcgcgc cacaaccttg agtgcctcac ccctcccacc ccgccccctt cctcccccct 180 ctgttgcttc cacaccttcc gcccctggga ggcagcatgg tcaagttttc caagacgctg 240 cacgtggccg cctcgaggtc gcgctgcttt gagctcctgc gcgactggtc gcgggcatcc 300 agctgggacc ccgccattgt cgagtcgtcc cggcaggcgg gccagccggc tgatgcgttt 360 ggcgccggga ccaagtggct catcatgttc aagcccagcc ccgacgccaa gcccatgtcg 420 gtggattata cgacgaccaa gttcgaggtg gaggaggcca agagcaccct cgtcttctct 480 ggaaacgcca cctttgtgcg gtccgtggac accctcgagt tgactgatgc ccccgcggcc 540 gacggcaccc cagggacaga cgtgaactac accgccgatg tgcggttgcg ctacctcctc 600 gctccatttt catttactct gcagggcaag atggatgaga tggctgagcc cgccatggag 660 ggtctgcaaa agttttgtga caaaaacttg ttgtgatggg gcggcagcgt gtaggcacag 720 ggaggcgcct gtgtcgtagc atgtacatat agtagatacg tcgtggacgg tccatgtgta 780 aagctgatat gacatgcata gg 802 <210> 2 <211> 159 <212> PRT <213> Pyropia tenera <400> 2 Met Val Lys Phe Ser Lys Thr Leu His Val Ala Ser Ser Ser Ser Arg   1 5 10 15 Cys Phe Glu Leu Leu Arg Asp Trp Ser Arg Ala Ser Ser Trp Asp Pro              20 25 30 Ala Ile Val Glu Ser Ser Gln Ala Gly Gln Pro Ala Asp Ala Phe          35 40 45 Gly Ala Gly Thr Lys Trp Leu Ile Met Phe Lys Pro Ser Pro Asp Ala      50 55 60 Lys Pro Met Ser Val Asp Tyr Thr Thr Thr Lys Phe Glu Val Glu Glu  65 70 75 80 Ala Lys Ser Thr Leu Val Phe Ser Ser Gly Asn Ala Thr Phe Val Arg Ser                  85 90 95 Val Asp Thr Leu Glu Leu Thr Asp Ala Pro Ala Ala Asp Gly Thr Pro             100 105 110 Gly Thr Asp Val Asn Tyr Thr Ala Asp Val Arg Leu Arg Tyr Leu Leu         115 120 125 Ala Pro Phe Ser Phe Thr Leu Gln Gly Lys Met Asp Glu Met Ala Glu     130 135 140 Pro Ala Met Glu Gly Leu Gln Lys Phe Cys Asp Lys Asn Leu Leu 145 150 155 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 tctagatggt caagttttcc aagacg 26 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 ggatccacaa caagtttttg tcacaaa 27 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 catatggtca agttttccaa gacg 24 <210> 6 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 gatatctcac aacaagtttt tgtcac 26

Claims (9)

delete A dry and osmotic stress resistance gene comprising the nucleotide sequence of SEQ ID NO: 1.
The method of claim 2,
The gene is a Pyropia- derived dry and osmotic stress resistance gene.
A dry and osmotic stress-resistant transformation vector comprising a gene according to claim 2 or 3.
A dry and osmotic stress-resistant transformant into which a transformation vector according to claim 4 is introduced.
The method of claim 5,
The transformant is an algae dry and osmotic stress resistant transformant.
The method of claim 5,
The transformant is a plant-derived dry and osmotic stress-resistant transformant.
A dry and osmotic stress resistant protein encoded by a gene according to claim 2.
A method according to claim 8, wherein the protein is used for the purpose of improving the drying or osmotic stress resistance of birds or plants.

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