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Kafe 바로가기Research in plant disease = 식물병연구, v.26 no.3, 2020년, pp.144 - 158
윤주연 (국립원예특작과학원 원예특작환경과) , 정재훈 (국립원예특작과학원 과수과) , 최승국 (농촌진흥청 연구정책국 연구운영과)
Quantitative reverse transcription PCR is used for gene expression analysis as the accurate and sensitive method. To analyze quantification of gene expression changes in apple plants, 10 housekeeping genes (ACT, CKL, EF-1α, GAPDH, MDH, PDI, THFs, UBC, UBC10, and WD40) were evaluated for their...
Quantitative reverse transcription PCR is used for gene expression analysis as the accurate and sensitive method. To analyze quantification of gene expression changes in apple plants, 10 housekeeping genes (ACT, CKL, EF-1α, GAPDH, MDH, PDI, THFs, UBC, UBC10, and WD40) were evaluated for their stability of expression during infection by Apple stem grooving virus (ASGV) or in cold-stress apple plant buds. Five reference-gene validation programs were used to establish the order of the most stable genes for ASGV as CKL>THFs>GAPDH>ACT, and the least stable genes WD40 * AI 자동 식별 결과로 적합하지 않은 문장이 있을 수 있으니, 이용에 유의하시기 바랍니다.
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표/그림 (8)
그림
Fig. 1. Range of Ct cycle threshold values for 10 apple candidate reference genes during infection by apple stem grooving virus (ASGV) or during incubation of three different cold conditions. The box plots show the cycle thresholds with median Ct value (central horizontal line in each box), the 25th and 75th percentile (boxes), and the ranges (whiskers) of the average of three independent experiments. The data were obtained from virus-free apple leaf (A), ASGV-infected apple leaf (B), apple bud incubated at 5°C (C), apple bud incubated at -8°C (D), or apple bud incubated at -15°C (E).
그림
Fig. 2. Expression stability of the 10 candidate reference genes analyzed using geNorm. The average expression stability values (M) are shown for the 10 candidate reference genes, ranking from least stable (left) to most stable (right), using data from the combination of three independent experiments for apple samples. The data show the pairwise variation (V) when the number of reference genes is increased from Vn/n+1 (V2/3, etc.), with a stability maximum of 0.15 permissible, for apple samples. The data were obtained from virus-free apple leaf (A), apple stem grooving virus (ASGV)–infected apple leaf (B), apple bud incubated at 5°C (C), apple bud incubated at -8°C (D), or apple bud incubated at -15°C (E). Data from the combination of three independent experiments, with the mean and standard error bars.
그림
Fig. 3. Expression stability of the 10 candidate reference genes analyzed using NormFinder. The average expression stability values are shown for the 10 candidate reference genes, ranking from least stable (left) to most stable (right), using data from the combination of three independent experiments for apple samples. The data were obtained from virus-free apple leaf (A), apple stem grooving virus (ASGV)-infected apple leaf (B), apple bud incubated at 5°C (C), apple bud incubated at -8°C (D), or apple bud incubated at -15°C (E). Data from the combination of three independent experiments, with the mean and standard error bars.
그림
Fig. 4. Expression and validation of eight candidate reference genes analyzed using delta Ct. (A) Range of Ct cycle threshold values for ten apple candidate reference genes. The box plots showing variation in ΔCt are as described in Fig. 1. The average expression stability values are shown, ranking from least stable (left) to most stable (right), using data from the combination of three independent experiment. The data were obtained from virus-free apple leaf (A), apple stem grooving virus (ASGV)-infected apple leaf (B), apple bud incubated at 5°C (C), apple bud incubated at -8°C (D), or apple bud incubated at -15°C (E). Data from the combination of three independent experiments, with the mean and standard error bars.
그림
Fig. 5. Relative expression levels of two target genes (MDP0000501598, MDP0000585239) during virus infection or cold stress using three normalized reference genes. The relative gene expression levels of the target genes from apple leaf samples (virus-free and apple stem grooving virus [ASGV]–infected) normalized to either two highly stable validated reference genes (CKL, ACT), or one less stable validated reference gene (WD40). The relative gene expression levels of the target genes from apple bud samples incubated at 5°C, -8°C, or 15°C, normalized to either two highly stable validated reference genes (WD40, CKL), or one less stable validated reference gene (ACT). The normalized gene expression in scale bar was presented as 1.0. Data was analyzed using two-way ANOVA and Duncan's multiple range test (P<0.05). Error bars indicate standard error of mean of three biological replicates.
표/그림 (8)
그림
Fig. 1. Range of Ct cycle threshold values for 10 apple candidate reference genes during infection by apple stem grooving virus (ASGV) or during incubation of three different cold conditions. The box plots show the cycle thresholds with median Ct value (central horizontal line in each box), the 25th and 75th percentile (boxes), and the ranges (whiskers) of the average of three independent experiments. The data were obtained from virus-free apple leaf (A), ASGV-infected apple leaf (B), apple bud incubated at 5°C (C), apple bud incubated at -8°C (D), or apple bud incubated at -15°C (E).
그림
Fig. 2. Expression stability of the 10 candidate reference genes analyzed using geNorm. The average expression stability values (M) are shown for the 10 candidate reference genes, ranking from least stable (left) to most stable (right), using data from the combination of three independent experiments for apple samples. The data show the pairwise variation (V) when the number of reference genes is increased from Vn/n+1 (V2/3, etc.), with a stability maximum of 0.15 permissible, for apple samples. The data were obtained from virus-free apple leaf (A), apple stem grooving virus (ASGV)–infected apple leaf (B), apple bud incubated at 5°C (C), apple bud incubated at -8°C (D), or apple bud incubated at -15°C (E). Data from the combination of three independent experiments, with the mean and standard error bars.
그림
Fig. 3. Expression stability of the 10 candidate reference genes analyzed using NormFinder. The average expression stability values are shown for the 10 candidate reference genes, ranking from least stable (left) to most stable (right), using data from the combination of three independent experiments for apple samples. The data were obtained from virus-free apple leaf (A), apple stem grooving virus (ASGV)-infected apple leaf (B), apple bud incubated at 5°C (C), apple bud incubated at -8°C (D), or apple bud incubated at -15°C (E). Data from the combination of three independent experiments, with the mean and standard error bars.
그림
Fig. 4. Expression and validation of eight candidate reference genes analyzed using delta Ct. (A) Range of Ct cycle threshold values for ten apple candidate reference genes. The box plots showing variation in ΔCt are as described in Fig. 1. The average expression stability values are shown, ranking from least stable (left) to most stable (right), using data from the combination of three independent experiment. The data were obtained from virus-free apple leaf (A), apple stem grooving virus (ASGV)-infected apple leaf (B), apple bud incubated at 5°C (C), apple bud incubated at -8°C (D), or apple bud incubated at -15°C (E). Data from the combination of three independent experiments, with the mean and standard error bars.
그림
Fig. 5. Relative expression levels of two target genes (MDP0000501598, MDP0000585239) during virus infection or cold stress using three normalized reference genes. The relative gene expression levels of the target genes from apple leaf samples (virus-free and apple stem grooving virus [ASGV]–infected) normalized to either two highly stable validated reference genes (CKL, ACT), or one less stable validated reference gene (WD40). The relative gene expression levels of the target genes from apple bud samples incubated at 5°C, -8°C, or 15°C, normalized to either two highly stable validated reference genes (WD40, CKL), or one less stable validated reference gene (ACT). The normalized gene expression in scale bar was presented as 1.0. Data was analyzed using two-way ANOVA and Duncan's multiple range test (P<0.05). Error bars indicate standard error of mean of three biological replicates.
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