The Functional Of Thioredoxin Genes In Arabidopsis Thaliana Under Environmental Stress

Plants frequently encounter stresses such as salinity, drought, heavy metal, radiation, low temperature, microbic infection; they adversely affect plant growth, development and productivity. The stability of plant internal redox environment and plant tolerance to stresses are closely related with a more efficient antioxidant and redox regulatory system. So it is helpful for us to study the response mechanism of oxidative stress, finding stress related genes and breeding for stress tolerance in crops.A. thaliana thioredoxins (Trxs) constitute a family of small and ubiquitous proteins with a highly conserved and prominent active site sequence WCG/PPC, that catalyse thiol-disulphide oxido-reduction. Plants possess two thioredoxin systems (FTR/Trx system and NTR/Trx system) that can be distinguished by the electron donor and the enzyme that catalyzes thioredoxin reduction. Thioredoxins play important roles in many plant physiological processes that include photosynthesis, signaling transduction, DNA synthesis, transcription, protein disulfide reduction, protein repair, filamentous phage assembly, cell apoptosis and seeds germinating and so on.In this study, the Arabidopsis thaliana Trx genes (AtTrx h1, AtTrx h3, AtTrx h5, AtTrx m1) were cloned from the green leaves by RT-PCR. And we did a particular research for the AtTrx h1, AtTrx h5 type genes.To understand the genes function in vitro, the Trx genes were cloned into the prokaryotic vector pQE-30, pQE-30-AtTrx h1, pQE-30-AtTrx h5 plasmid was transformed into E. Coli M15. After the inducement with IPTG, the His-AtTrx h1 and His-AtTrx h5 fusion protein were finally purified by Ni⁺-sepharose affinity column and analyzed by SDS-PAGE. The recombinant His-AtTrx h1, His-AtTrx h5 protein activity were measured with insulin and DTT. The results suggested the AtTrx h5 have a higher activity than AtTrx h1. In order to investigate AtTrxs' relationship with abiotic stress in protein level, we prepared the polyclonal antibody with recombinant AtTrx h1 and AtTrx h5 protein.To understand the responsive relationship between Trxs and environmental stress, the transcription expression profile of Trxs was studied by Northern blot analysis using A. thaliana suspension cells under several stresses. The results showed that the accumulation of Trx mRNA was observed in salts (NaCl, NaHCO₃), ABA, and H₂O₂ conditions, and the intensity of stresses influenced the expression of Trxs. These results suggested that the expression of Trxs response to several abiotic stresses, but the response mechanisms are different.To further investigate the role of the Trxs tolerance, AtTrx h1, AtTrx h5 and AtTrx m1 were cloned into pBI121, and introduced into A. thaliana by Agrobacterium tumefaciens-mediated transformation using the vacuum infiltration method. The independent and stable expression lines of over-expressing different Trxs were confirmed by PCR, Northern and Western analysis. Salt tolerance of the independent transgenic lines in germination was examined, and the wide type plants were restrained more heavily than the the transgenic plants under NaCl stress. For treatment with 100 mM NaCl, the root length of AtTrx h1 and AtTrx h5 increased about 19% and 77% respectively; for treatment with 100 mM NaCl, the root length ofAtTrx h1 and AtTrx h5 increased about 14% and 45%; and under 200mM NaCl, the wide type could't germinate completely, but the trasnsgenic plants harboring CaMV35S: AtTrx h5 could germinate though the growth was hampered. In oreder to confirm the relationship between AtTrx genes with the salt stress in the development of the plants, the transplant experminents were done under salt stress. The results indicated that the salt tolerance of the transgenic plants increased and the transgenic plants grown better than the control. Under 100 mM NaCl stress, the growth of the wide type plants were restrained and the leaves showed some chlorosis and decreased vigor, but the transgenic plants harboring AtTrx h5 and AtTrx h1 type both have a higher resistance, especially the transgenic plants harboring AtTrx h5. It is according with the results of the trx assays that AtTrx h5 have a stronger resistance than the AtTrx h1.The research suggested that A. thaliana thioredoxin family genes play an important role in abiotic stress tolerance and can enhance the transgenic plants' resistance to salt stress.