Expression of Antioxidant Enzyme Genes in Rice under PEG-simulated Drought-stress
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摘要:目的 干旱是影响水稻生产的重要环境因素之一，在干旱条件下水稻植株体内会发生一系列的抗逆反应，其中参与防御反应的关键酶基因表达会发生明显的变化。因此，本研究拟分析干旱胁迫处理后抗氧化酶类基因的表达变化，为进一步研究水稻抗旱机制提供理论参考。方法 采用质量体积比为0（CK）、18%、20%、22%、24%、26%的聚乙二醇（PEG6000）对三叶一心期的籼稻航2号植株进行干旱胁迫处理，筛选适合处理籼稻航2号的PEG6000质量体积比；进一步采用PEG6000对航2号植株进行干旱胁迫处理，分别于处理0、2、4、8、12、24、48、72 h取样；并用SYBR Green I荧光定量PCR（qRT-PCR）分析PEG6000处理不同时间段后植株中抗氧化酶类基因表达，包括过氧化氢酶（CATA、CATB、CATC）、过氧化物酶（POX5.1、POX1）、超氧化物歧化酶（plastidic Cu/Zn-SOD，cytosolic Cu/Zn-SOD）、抗坏血酸过氧化物酶（APX）、谷胱甘肽还原酶（GR）基因的表达变化。结果 根据表型观察和植株存活率，筛选出籼稻航2号对PEG6000的耐受临界质量体积比为22%；qRT-PCR结果表明PEG6000胁迫处理后9个基因的表达均出现上调，大部分基因表达都呈先上调后下调的趋势，且一般PEG处理4 h之后基因表达出现较明显上调，说明这些基因均不同程度地参与了PEG胁迫反应；其中，过氧化氢酶A基因（CATA）表达变化最显著，处理8 h表达量上调至处理0 h的28倍。结论 PEG6000胁迫处理后主要的抗氧化酶类基因表达发生了明显的变化。
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关键词:
- 水稻 /
- 聚乙二醇(PEG6000) /
- 干旱胁迫 /
- 抗氧化酶基因 /
- 表达分析
Abstract:Objective Expression of antioxidant enzyme genes of rice in response to drought-stress was studied.Method Simulated drought conditions using PEG6000 on Indica rice Hang 2 were used for the experimentation. The plants at 3-leaf stage were initially treated with 0% (CK), 18%, 20%, 22%, 24% or 26% PEG6000 to determine the appropriate concentration for the subsequent test. Under the selected PEG6000 treatment level, plant samples were collected at 0, 2, 4, 8, 12, 24, 48 and 72 h for analysis. The expressions of antioxidant enzyme genes (i.e., CATA, CATB and CATC), peroxidase genes (i.e., POX5.1 and POX1), superoxide dismutase genes (i.e., plastidic Cu/Zn-SOD and cytosolic Cu/Zn-SOD), ascorbate peroxidase gene (i.e., APX), and glutathione reductase gene (i.e., GR) of the rice plants were determined by qRT-PCR.Result Based on the phenotype and survival rate of the rice plants in the preliminary test, 22% PEG6000 was chosen for the simulation experiment. The results of qRT-PCR showed that all 9 genes were upregulated initially under the treatment but downregulated afterward. Most of the genes significantly upregulated 4 h after treatment showing a response of the genes to the stress. In particular, CATA exhibited a most significant change at 8 h which was 28 times of that at 0 h.Conclusion The expression of antioxidant enzyme genes significantly reacted to the PEG6000 treatment.-
Keywords:
- rice /
- PEG6000 /
- drought stress /
- antioxidant enzymes genes /
- expression analysis
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图 1 不同浓度PEG6000处理航2号植株情况
注:每幅图从左到右营养液中PEG6000的质量体积比分别为0%(CK)、18%、20%、22%、24%、26%；处理时间A: 0 h，B: 3 h，C: 24 h，D: 48 h，E: 72 h，F: 96 h，G: 7 d，H:恢复生长8 d。
Figure 1. Hang 2 plants treated by varying concentrations of PEG6000
Note: PEG600 concentrations in photos from left to right are 0% (CK), 18%, 20%, 22%, 24% and 26%; treatment time A=0 h, B=3 h, C=24 h, D=48 h, E=72 h, F=96 h, G=7 d, H=recovery after 8 d.
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图 4 抗氧化酶基因的RT-PCR扩增
注：pla SOD为plastidic Cu/Zn-SOD; cyt SOD为cytosolic Cu/Zn-SOD
Figure 4. RT-PCR amplifications of antioxidant enzyme genes
Note: pla SOD:plastidic Cu/Zn-SOD; cyt SOD:cytosolic Cu/Zn-SOD
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图 5 qRT-PCR分析抗氧化酶基因的表达情况
注：A为过氧化氢酶A基因表达量，B为过氧化氢酶B基因表达量，C为过氧化氢酶C基因表达量，D为过氧化物酶5基因表达量，E为过氧化物酶1基因表达量，F为质体铜/锌超氧化物歧化酶基因表达量，G为细胞质铜/锌超氧化物歧化酶基因表达量，H为抗坏血酸过氧化物酶基因的表达量，I为谷胱甘肽还原酶基因的表达量。
Figure 5. Expression analysis on antioxidant enzyme genes by qRT-PCR
Note:A:The relative expression of CATA, B:The relative expression of CATB, C:The relative expression of CATC, D:The relative expression of POX 5.1, E:The relative expression of POX 1, F:The relative expression of plastidic Cu/Zn-SOD, G:The relative expression of cytosolic Cu/Zn-SOD, H:The relative expression of APX, I:The relative expression of GR.
表 1 抗氧化酶基因引物序列
Table 1 Primers of antioxidant enzyme genes
基因名称
Gene names上游引物F/下游引物R(5′-3′)
Upstream primer F/Downstream primer R产物大小
Product/bp过氧化氢酶A基因CATA F：GAGGAGGCAGAAGGCGACGATA
R：CCCCCAACGACTCATCACACTG194 过氧化氢酶B基因CATB F：GACGGATGGTCCTGAACAAAAACA
R：CAAGACGGTGCCTTTGGGTATCA159 过氧化氢酶C基因CATC F：CTTCCCCGTCTTCTTCATCCGC
R：TCGTCGAAGAGGAAGGTGAACAT159 过氧化物酶5基因POX5.1 F：ACTTGGTTGCTCTCTCAGGTGCG
R：GGTGGGCGTCGTCGTGTC182 过氧化物酶1基因POX1 F：ACTCGTGCCCCAAGGCGAAGGA
R：GCTGTTGTCCAGGAGCACAGACG149 质体铜/锌超氧化物歧化酶基因plastidic Cu/Zn-SOD F：CCACCTCCACGAGTTTGGCGAT
R：CTCAGCTACACCTTCAGCATTGGC154 细胞质铜/锌超氧化物歧化酶基因cytosolic Cu/Zn-SOD F：GGAAATGTCACCGCTGGAGAAG
R：AACGACGGCTCTGCCAATGATT102 抗坏血酸过氧化物酶基因APX F：CTGCCGTCCCCTTCCACCCA
R：CCGCCAGAGAGGGCAACAAT154 谷胱甘肽还原酶基因GR F：TTCCTCCAAAGCCTGCTGTTCACT
R：GCCAGCCAACTAAACCTGATTACA101 内参基因，真核起始因子eIf4a F：TTGTGCTGGATGAAGCTGATG
R：GGAAGGAGCTGGAAGATATCATAGA76 内参基因，肌动蛋白基因Actin150 F：AGTGTCTGGATTGGAGGAT
R：TCTTGGCTTAGCATTCTTG150 
下载: 导出CSV
表 2 不同质量体积比的PEG6000处理后植株存活率
Table 2 Plant survival rates after PEG6000 treatments in different mass and volume ratios
PEG质量体积比
Mass and volume
ratio of PEG/%植株数量
Plant
number存活的
植株数量
Survival plant存活率
Survival
rate/%0 25 25 100 18 25 25 100 20 25 25 100 22 25 3 12 24 25 0 0 26 25 0 0
下载: 导出CSV
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