| Thioredoxins, a ubiquitous heat-stable, acidulous small proteins with a redox activedisulfide bridge in its conserved motif -CP(G)PC-, distribute universally in eukaryote andprokaryote and have a molecular mass of approximately 12Kda. They are a sort of redoxregulation proteins, which play an important role in regulating various physiological reactionsrelated to redox status in cells. By the disulfide/dithiol interchange reaction, Thioredoxins cantransmit electrons and protons, therefore can transmit the regulatory redox signals to selectedtargets (enzymes, proteins, transcription factors etc) and effect many processes.Plants have a more complex thioredoxin system than do animals and prokaryote. Theydistribute widely in cytoplasm, chloroplast and mitochondria, which facilitate theirinvolvement in modulating diverse life activities. Thioredoxins in chloroplast act in theelectron transport chain in photosynthesis while Thioredoxins in cytoplasm might involve inthe cellular redox regulation, which can be disturbed by oxidative stress. As is known,oxidative Stress is caused ubiquitously by various stresses. It can alter the stable redoxcellular environment which is essential for normal plant physiological metabolism, damagethe cell directly and affect the normal metabolism, and finally lead to serious stress status,sometimes even cause death.Thellungiella halophila is close relative to Arabidopsis which is also belong toCruciferae and has good genetic features such as similar morphology, small genome size,short life cycle, high seed number and an efficient transformation method. However,Thellungiella halophila is able to withstand dramatic salinity shock up to 500mM NaCl. Thisplant does not produce salt glands or other complex morphological alterations either before orafter salt adaptation. Therefore, the study of function of thioredoxin from Thellungiellahalophila is quite significant for expatiating plant response mechanism of oxidative stress,understanding the metabolism alteration and adjustment of plant under stresses andexpounding the stress tolerance metabolism.A ThTRX h gene was obtained from ESTs (expressed sequence tags) acquired fromλZap-cDNA library of Thellungiella halophila treated with 200mmol.L-1NaCl in our lab.Expression in vitro and activity experiment were performed and the result proved thatThTRXh did have TRXh activity. Since family members of TRXh exist not only in cytoplasmbut also in Endoplasmic reticulum and mitochondria, and different localizations affect theirpotential functions, we first identified the subcellular localization of ThTRXh in order tounderstand its biological function. By constructing the GFP transient expression vector ofThTRXh and transforming it into the Arabidopsis mesophyll protoplasts, ThTRXh wasinitially localized in cytoplasm by laser scanning confocal microscope. To further verify theresult, a GFP Plant Binary Expression Vector of ThTRXh was constructed at the same time.This vector was transformed into the onion epidermal cells by agrobacterium-mediatedtransient transformation and the scanning result of transformed cells showed that ThTRXh didlocalize in the cytoplasm. Both the subcellular localization results were in accordance with theresult of iPSORT software. AtTRXh mutant were ordered from the salk library and thephenotype of the mutant was analyzed to set the foundation for the following work of mutantphenotype complementation by transforming ThTRXh into the mutant. Our results showedthat the germination rate of mutant was much lower than that of the wild type, but theseedling growth of mutant and the root growth rate were both better. From the directlocalization of H2O2 in leave, it can be seen that the mutant plant leaves accumulate muchmore H2O2 compared with that of the wild type plant, which reveals that the mutant suffersmore severe oxidative stress. These results demonstrated that thioredoxin h involved in seedgermination and seedling growth. It might act as a kind of massager in plant oxidative stressresponse.
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