[논문]Heterologous expression of the Arabidopsis DREB1A/CBF3 gene enhances drought and freezing tolerance in transgenic Lolium perenne plants

Li, Xue; Cheng, Xiaoxia; Liu, Jun; Zeng, Huiming; Han, Liebao; Tang, Wei

doi:10.1007/s11816-010-0157-9

Heterologous expression of the Arabidopsis DREB1A/CBF3 gene enhances drought and freezing tolerance in transgenic Lolium perenne plants 원문보기

Plant biotechnology reports, v.5 no.1, 2011년, pp.61 - 69

Li, Xue (Institute of Turfgrass Science, Beijing Forestry University) , Cheng, Xiaoxia (Institute of Turfgrass Science, Beijing Forestry University) , Liu, Jun (Institute of Turfgrass Science, Beijing Forestry University) , Zeng, Huiming (Institute of Turfgrass Science, Beijing Forestry University) , Han, Liebao (Institute of Turfgrass Science, Beijing Forestry University) , Tang, Wei (College of Horticulture and Gardening, Yangtze University)

Abstract ▼ AI-Helper

The dehydration-responsive element binding proteins (DREB1)/C-repeat (CRT) binding factors (CBF) function as transcription factors and play an important role in agricultural biotechnology and molecular biology studies of drought and freezing stress tolerance. We generated transgenic Lolium perenne plants containing the PCR-cloned Arabidopsis DREB1A/CBF3 gene (AtDREB1A/CBF3) to study the function of this gene construct in drought and freezing tolerance in a species of turfgrass. Compared to the control, AtDREB1A/CBF3 transgenic L. perenne plants showed enhanced drought and freezing stress tolerance. The activities of the enzymes superoxide dismutase (SOD) and peroxidase (POD) were higher in transgenic plants than in the non-transgenic plant control. These results demonstrate that the expression of the AtDREB1A/CBF3 gene in transgenic L. perenne plants enhanced drought and freezing tolerance and that the increased stress tolerance was associated with the increased activities of antioxidant enzymes. These results are relevant to stress biology and biotechnology studies of turfgrass.

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For the northern blot analysis, total RNA was extracted from leaves of transgenic perennial ryegrass plants using TRN201 RNA Purification Reagent (Dp405-02; Tiangen Biotech). After 5 lg of total RNA were separated by electrophoresis, RNAs were blotted onto a membrane (Millipore, Bedford, MA), and hybridization was performed using the DIG High Prime DNA Labeling and Detection Starter kit I [nitro-blue tetrazolium/2,6-dichloroindophenol (NBT/BCIP); Roche Diagnostics]. The labeling of CBF3 probes, hybridization, and washing of the membrane were performed according to the manufacturer’s protocol.
The GUS-expressing vector was used in this study to monitor the transformation rate. To establish a stable genetic transformation system, we constructed an expression vector carrying the HPT and chimeric GUS coding sequences under the control of the CaMV35S promoter and the CBF3 gene under the control of UBI and then used this vector for Agrobacterium-mediated transformation.

대상 데이터

4 Physiological changes in transgenic plants derived from transgenic lines L3 and L9, respectively, including effects of the drought stress on electrolyte leakage (a), malondialdehyde (MDA) content (b), peroxidase (POD) activity (c), superoxide dismutase (SOD) activity (d) of transgenic perennial ryegrass plants and control plants. Experiments were repeated three times, and each replicate consisted of three plants. Data represent the mean ± standard deviation

데이터처리

Data obtained from the different experiments were analyzed using the General Linear Model (GLM) procedure of SAS (SAS, Cary, NC), employing analysis of variance (ANOVA) models. Differences between mean values obtained from at least three independent experiments were assessed with the least significant difference test at a significance probability level of 5%.

성능/효과

Data obtained from the different experiments were analyzed using the General Linear Model (GLM) procedure of SAS (SAS, Cary, NC), employing analysis of variance (ANOVA) models. Differences between mean values obtained from at least three independent experiments were assessed with the least significant difference test at a significance probability level of 5%. Each value was presented as the means ± standard error of the mean (SEM), with a minimum of three replicates.
In conclusion, our results suggest that AtDREB1A/CBF3 may be a regulator that is sufficient on its own to control the gene expression involved in antioxidant enzyme biosynthesis in transgenic L. perenne plants. Enhanced drought and freezing tolerance was associated with increased activities of antioxidant enzymes, and this result may prove to be useful in stress biology studies of turfgrass.
9% at infection times of 10 and 20 min, respectively. Our results show that an Agrobacterium concentration at OD₆₀₀ = 0.1, infection period of 20 min, and co-cultivation time of 4 days were the best conditions for A. tumefaciens-mediated genetic transformation in L. preened (data not shown).
Survival rates of plants derived from transgenic cell lines L3, L5, L9, L12, L15, L18, and L19 and of non-transgenic control were determined 6 weeks after the dehydration treatments followed by re-watering for drought treatment and 6 weeks after plants were exposed to -6°C for 1 day (freezing treatment) followed by a return to 25°C.
The results of our study show that overexpression of the transcription factor AtDREB1A/CBF3 gene enhances drought and cold tolerance in transgenic L. perenne plants. Transgenic plants harboring a corn ubiquitin promoter driving the AtDREB1A/CBF3 gene had higher SOD and POD activities than the wild-type controls.
Survival rates of plants derived from transgenic cell lines L3, L5, L9, L12, L15, L18, and L19 increased up to 11-fold compared to the non-transgenic control 6 weeks after the freezing treatment (exposure of transgenic plants to -6°C for 1 day) followed by a return to 25°C (Table 1). These results showed that heterologous expression of the Arabidopsis DREB1A/CBF3 gene enhances drought and freezing tolerance in transgenic L. perenne plants.

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