拟南芥活性氧不敏感型突变体的筛选与特性分析

Isolation and characterization of reactive oxygen species-insensitive mutants in Arabidopsis thaliana

采用 EMS化学诱变方法与 H2 O2 氧化胁迫选择 ,以根在重力作用下的弯曲生长为指标 ,筛选得到拟南芥活性氧不敏感型突变体。对突变体杂交后代遗传分析表明 ,突变株对活性氧不敏感性状为隐性单基因突变所致 ;生理生化分析表明突变体对 H2 O2 有很强的抗性 ,表现为气孔开度对 H2 O2 不敏感和 H2 O2 胁迫时较低的膜脂过氧化水平。运用 L SCM技术并结合 H2 O2 荧光探针 H2 DCFDA检测外源 ABA诱导保卫细胞内产生 H2 O2 的情况 ,结果显示突变体体内荧光强度比对照低 ,暗示了突变体体内消除 H2 O2 的能力可能有所提高 ,增强了植株对氧化胁迫的抗性。拟南芥活性氧不敏感突变体的筛选 ,不仅为人们深入研究活性氧在细胞内的作用提供良好的实验材料 。

Isolation of the putative reactive oxygen species-insensitive mutants (\%ros\%) and characterization of mutants were performed by using root-bending assay and genetic analysis in Arabidopsis thaliana.10 -4 mol/L H 2O 2 was established for screening-concentration based on the effects of H 2O 2 on root growth and stomata aperture.200 000 ethylmethane sulfonate (EMS)-mutagenized Arabidopsis M 2 seeds were screened for reactive oxygen insensitive mutants,using root-bending assay on 10 -4 mol/L H 2O 2 agar plates,of twenty-six reactive oxygen species-insensitive mutants were obtained.Genetic analysis showed that mutations are recessive and are allelic to each other.We directly examined the production of H 2O 2 in guard cells in wild-type and mutant by using LSCM based on 2,7-dichlorofluorescin diacetate.The results indicated that the mutation of \%ros\% mutant was possibly related to the scavenging enzymes of \%ros\%,which abolished the unwanted H 2O 2 to avoid the damage of cell.The acquisition of reactive oxygen species-insensitive mutant not only offers perfect material for unraveling the molecular mechanisms of reactive oxygen species generation in plant cells under various environmental stresses,but also provides new insights into oxidative signal transduction in plant cells.