A Set Of Promoters Activated By Stress Factors In Arabidopsis Thaliana And Study Of Their Application

The ability to switch from one physiological pathway, or even developmental program to another in response to an environmental signal is crucial for the survival of all organisms. This is particularly true in plants, in which, a high degree of developmental plasticity is required for plants to cope with their sessile lifestyle. This means that plants, unlike most animals, are unable to move away from an unfavorable environment. They must, instead, cope with the stress in situ, by producing protective compounds, altering their morphology to adjust to the stress and, in some more extreme cases, by accelerating their life cycle to survive in the form of seeds. Before that, plants must have changed their gene expression pattern to respond to the environmental agent. To response environmental stress, the expression of stress resistance genes in plant are regulated by inducing regulative promoters. Namely: When the plants are stressed by an environmental stresss factor, those stress-related gene will be activated; when stress disappeares, those genes will be turned off. While it is very important to learn the mechanism of regulated expression of plant genes, these stress-inducible promoters could be widely applied in plant genetic engineering. This study is to identify promoters that are activated by stress factors; to analyze their regulation mechanisms; to engineer these stress-inducible genes for practical applications in basic research and applied sciences.This study has accomplished the following major milestones:1.We have successfully applied a novel PCR-based differential display technology in plant research. This technology is based on annealing control primers (ACPs) to identify up-regulated (or down-regulated) genes of Arabidopsis by Cd2+ exposure. Nineteen differentially expressed transcripts bands were isolated and sequenced. They represent eighteen unique genes. Among them, six genes were further confirmed by RT-PCR that they were significantly induced by cadmium treatment. They include LEA(late embryogenesis abundant protein), AtGSTF2 (Glutathione S-transferase 2), AtGSTF6(Glutathione S-transferase 6), AtHsp70(heat shock protein 70), AtsHspl7.6-C1(17.6 kDa classl small heat shock protein) and AtsHsp17.6-C2(17.6 kDa class2 small heat shock protein). These results will help us to understand detoxification mechanism of plant to cadmium.2. Among six genes mentioned above, three heat shock protein genes were undetectable in vegetative tissues under normal growth condition, but their expression levels are increased 9-16 times under cadmium exposure. They also can be induced by other heavy metal ions, such as Ni+,Pb2+,Cu2+,Zn2+, Al3+, and so on. Heat-shock, osmotic and oxidative stress can also induce their expression. Thus, they appears to play major role in maintaining cell function and survival during stress or facilitating recovery from stress. Their promoters are characterized as strongly heat-inducible and multiple-stress responsive, which can be used in plants genetic engineering. By over-expressing the AtsHsp17.6-C2 gene in Arabidopsis thaliana, the transgenic plants have acquired stress-tolerance.3. We constructed a high efficient inducible expression system in plants, composed of the heat-shock plant promoter of AtsHsp17.6-C2 from Arabidopsis thaliana. Expression profiles of reporter gene fused to this promoter during and after heat-shock treatment of plants were investigated. The AtHsp17.6-C2 promoter presents a weak basal expression at a growth temperature of 22℃. Their expression levels were strongly induced by incubation at 34-37℃for 2h. The optimal inducing temperature was 37℃and GUS activity increased about 80 folds. This temperature was also acclimation temperature for thermo-tolerance test to Arabidopsis and may do less harm to the plant. Because of the temperature responsive feature of this promoter, we can modulate induction activity of AtHsp17.6-C2 promoter by controlling heat-shock temperature and duration. The GUS specific activity reached maximum levels at the 5 h after a single dose heat-shock treatment and then began to decrease. However, it still maintains a 73% of the highest GUS specific activity at 24 h after heat-shock treatment. Multiple heat-shock treatments did not affect plant growth and the GUS activity was still repeatedly induced effectively. So we can get a relatively stable expression levels through repetitions of heat-shock treatment.Major accomplishments of this study:1. We established a platform for a novel differential display PCR method. This method is based on annealing control primers (ACPs) to identify DEGs (different expressing genes, DEGs) under different physiological conditions. This is very useful for study of gene function and in application for scientific research and plant gene engineering.2. We demonstrated that over-expressing AtsHspl7.6-C2 can improve plant stress tolerance in Arabidopsis thaliana.3. A high efficient inducible gene expression system in plants was described, composed of the heat-shock plant promoter of AtHspl7.6-C2 from Arabidopsis. Since all plants may have a highly conserved response to elevated temperature, this system should be applied conveniently in other kinds of plants.