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Research Article

Overexpression of Oshsp16.9 Gene Encoding Small Heat Shock Protein Enhances Tolerance to Abiotic Stresses in Rice

Plant Breeding and Biotechnology 2014;2(4):370-379.
Published online: December 31, 2014

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*Corresponding author: Kwon Kyoo Kang, kykang@hknu.ac.kr, Tel: +82-31-670-5104, Fax: +82-31-670-5100
• Received: December 22, 2014   • Revised: December 26, 2014   • Accepted: December 26, 2014

Copyright © 2014 The Korean Society of Breeding Science

This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Overexpression of Oshsp16.9 Gene Encoding Small Heat Shock Protein Enhances Tolerance to Abiotic Stresses in Rice
Plant Breed. Biotech.. 2014;2(4):370-379.   Published online December 31, 2014
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Overexpression of Oshsp16.9 Gene Encoding Small Heat Shock Protein Enhances Tolerance to Abiotic Stresses in Rice
Plant Breed. Biotech.. 2014;2(4):370-379.   Published online December 31, 2014
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Overexpression of Oshsp16.9 Gene Encoding Small Heat Shock Protein Enhances Tolerance to Abiotic Stresses in Rice
Image Image Image Image Image Image
Fig. 1 A, Sequence alignment of molecular chaperones, including the α-crystallin domains (ACDs) from different proteins. The α-crystallin domains (ACD) were marked by underline. B, Sequence alignment of the known members of the HSP family. Accession numbers are as follows: OsHSP16.9, AK105464, AK072830 and AK061896 (Oryza sativa Japonica group); HQ156231 (Oryza sativa Indica group); EU952538 (Zea mays); GU120339 (Oryza sativa Japonica group); X13431 (Triticum aestivum); X94192 (Cenchrus americanus); BAB02269 and NP200076 (Arabidopsis thaliana).
Fig. 2 A, Cytoplasmic localization of the Oshsp16.9 gene. p35S:GFP-Oshsp16.9 was transiently expressed in onion epidermal cells. The photograph was taken in the dark field for green fluorescence (a), and under bright light for the morphology of the cell (b). B, Southern blot analysis. The specific region of Oshsp16.9 was used as a probe. Restriction enzymes used were as follows; M: molecular marker, B: BamHI, E: EcoRI, H: HindIII, P: Pst I. C, Relative expression of Oshsp16.9 in organs collected from rice. Data presented are means and standard deviations of 3 independent replicated experiments.
Fig. 3 Expression profiles of Oshsp16.9 in shoots and roots of rice after abiotic stress treatments. Expression levels were examined via real-time PCR analysis at various times after treatment with salt (250 mM NaCl), heat (45°C), cold (4°C), 100 μM abscisic acid (ABA), 300mM mannitol and osmotic stress (15% PEG6000) in tissue from 20-day-old plants. Actin was used as a positive control for relative expression for each treatment. Data presented are means ± SD of three independent replicated experiments.
Fig. 4 Expression levels of Oshsp16.9-overexpressing lines. A: PCR amplification of transferred Oshsp16.9 genes in transgenic rice lines. The amplification products were separated using a 1% agarose gel. Lane M: 1-kb DNA ladder; Lane WT: wild-type plant; Lanes T1–T9: PCR products generated from the DNA template of independent transgenic lines; and Lane P: PCR product of the vector plasmid that contained Oshsp16.9. B: Southern blot analysis for detection of the Oshsp16.9 gene in transgenic plants. Genomic DNA of five transgenic lines were digested with EcoRI, separated with 0.8% agarose gel electrophoresis and hybridized with a Oshsp16.9 fragment probe. C: Analysis of Oshsp16.9 gene expression in the transgenic rice lines by RT-PCR. As a loading control, the samples were also amplified with specific primers for the rice actin gene.
Fig. 5 A, Drought and salt tolerance assays of Oshsp16.9 transgenic rice. Three-week-old seedlings were exposed to air conditions for 10 h, and then transferred to normal hydroponic conditions and allowed to recover for 20 days. In addition, three-week-old seedlings were watered with 250 mM NaCl solution for 24 h, and then allowed to recover for 20 days. B, Survival rates of the WT and transgenic rice lines. The data presented are the means ±SE of three independent experiments. The values with significant differences according to t-tests are at the level of P < 0.05.
Fig. 6 Relative expression levels of candidate genes in Oshsp16.9-overexpressing lines under drought (exposed to air) and salt stresses (250 mM NaCl). Total RNA was extracted from three-week-old rice seedlings.
Overexpression of Oshsp16.9 Gene Encoding Small Heat Shock Protein Enhances Tolerance to Abiotic Stresses in Rice