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    Functional expression of Vitreoscilla hemoglobin (VHb) in Arabidopsis relieves submergence, nitrosative, photo-oxidative stress and enhances antioxidants metabolism

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    Abstract
    A chimeric construct consisting of the double CaMV35S promoter fused to the Myc-vhb gene encoding Vitreoscilla (bacterial) hemoglobin (VHb) was introduced into Arabidopsis thaliana and transgenic plants were cultured under various growth and stress conditions with the aim of studying the generality phenomena. Molecular analysis indicated that the exogenous vhb was stably integrated into the Arabidopsis genome and successfully expressed. Constitutive vhb expression improved the germination rate of transgenic seeds. Under submergence conditions, in vitro and in vivo bioassays revealed that the vhb expression protected transgenic plants against prolonged waterlogging. Leaf discs from the transgenic plants maintained in standard growth conditions reveal increased tolerance to nitrosative stress induced by sodium nitroprusside (SNP) treatment. Significant differences in chlorophyll a, b, total protein content and conductivity were observed among the transgenic, wild type (WT) and blank transformation (CK) plants. No significant differences were observed between the tested VHb, WT and CK lines in the presence of oxidative stresses by paraquat treatment. VHb expression also affected the levels of some Arabidopsis endogenesis genes that are involved in oxygen metabolism and antioxidants biosynthesis; up-regulating their expression at transcriptional level. LC–MS revealed that VHb expression shifted plant secondary metabolism, which may reflect the availability of extra metabolite production for possible enhanced protection ability. VHb transgenic plants were more tolerant to photo-oxidative damage due to the increased antioxidants, such as ascorbate. These results suggested that vhb gene might have the potential use in molecular breeding for the improvement of stress resistance and fine chemicals production in plants.
    Article Outline
    1. Introduction
    2. Materials and methods
    2.1. Construction of expression cassette, transformation, and growth of plants
    2.2. DNA extraction and PCR analysis
    2.3. Southern blot analysis
    2.4. Protein expression analysis
    2.5. Growth evaluation and stomatal aperture measurements
    2.6. Submerged culture and waterlogging tolerance assay
    2.7. Analysis of oxidative and nitrosative stress tolerance
    2.8. Measurement of chlorophyll content, total protein and electrolyte leakage
    2.9. Real-time quantitative analysis
    2.10. LC–MS analyses of secondary metabolites and compounds identification
    2.11. Statistical analysis
    3. Results
    3.1. Plant transformation and molecular characterization of transgenic Arabidopsis
    3.2. Plant growth performances
    3.3. Plant responses to submergence under in vitro and in vivo submerged conditions
    3.4. Assessment of in vitro nitrosative and oxidative stress tolerance
    3.5. Effect of VHb-transgene on plant endogenous gene expression and secondary metabolism profiles
    4. Discussion
    Acknowledgements
    References

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    作者:Wang, Zinan, Xiao, Ying, Chen, Wansheng, Tang, Kexuan, Zhang, Lei 来源:Elsevier 发布时间:2011年07月13日