Studies On The Interaction Of Calmodulin And Calcineurin Induced By Aluminum As Well As Their Resistance To Aluminum

Aluminum toxicity in acidic soils is one of the limiting factors affecting crop yield. Typically, the solution of aluminum toxicity is a lot of lime to raise the pH of the soil, results to the free aluminum precipitation. However, this method is difficult to solve the problem of soil acidity and aluminum toxicity, but also there is a potential environmental problem. The effective and sustainable measures of improving crop yields on acid soils are screened and nurtured crop varieties resistant to aluminum toxicity. Therefore, the study of the mechanism of resistance to aluminum toxicity, cloning Al tolerance gene can lay the theoretical foundation for the genetic improvement of Al-sensitive plants.In acidic soils, aluminum also produce toxic effects on microorganisms. Study the mechanisms of aluminum toxicity and resistance on microorganisms, can be resistant to study the mechanism of aluminum toxicity to plants to provide a new perspective. Microorganisms grow fast, short, easy to operate, is the study of the mechanism of resistance to aluminum better model organism. In this paper, separated by Yunnan tea acidic soils Cryptococcus humicola as research material, its resistance to aluminum concentrations up to 200mM. Calmodulin ubiquitous in eukaryotic cell, is a signal component of a variety of biological effects. Calcium/calmodulin pathway is composition of calcium ions, calmodulin and the downstream target proteins, through the action of the target proteins to transmit signals and play a biological function. Studies have shown that calcium, calmodulin and calmodulin-binding proteins involved in abiotic including drought, salt stress, cold and heat shock, including a stress response. According to SSH cDNA library in response to aluminum stress of the C. humicola and CaM gene as aluminum processing time expression profiling data, we speculate that CaM-mediated signaling pathways may be involved in aluminum stress at the molecular level. Therefore, we intend to Cryptococcus neoformans as a model organism, isolated and identified CaM-binding protein, CaM and studied its downstream components the role and function of the stress-resistant aluminum.1. Calmodulin and its downstream target proteins-calcineurin and calmodulin kinase inhibitors affect on the growth of C. humicola. The results showed that adding FK-506 and KN-93in control medium, the growth of C. humicola is similar as control, and add TFP, growth was significantly inhibited. Add in a medium containing Al FK-506 and TFP growth was inhibited, and added KN-93 did not affect the growth of basic income. So ba we only created transgenic calmodulin and calcineurin in yeat.2. Impact of calmodulin and its downstream target proteins----calcineurin and calmodulin kinase inhibitor on C. humicola resistant aluminum. To further validate the role of calmodulin and calcineurin plays in the growth and aluminum tolerance in. humicola, On each agar plate not containing aluminum-containing inhibitors, and growth of colonies was observed in the presence of C. humicola 50mM solid aluminum plate and inhibitor. The results show that it does not contain aluminum containing calmodulin inhibitor (TFP), calcineurin inhibitors (FK-506) and calmodulin kinase inhibitor (KN-93) solid plates native yeast colonies with Cryptococcus increased dilution, compared with CK only added to the solid plates calmodulin inhibitor (TFP) of yeast colonies smaller, growth was significantly inhibited. And to add another two inhibitors (calcineurin inhibitor (FK-506) and calmodulin kinase inhibitor (KN-93)) of yeast colonies compared with CK. no significant difference, which the image above the results are consistent with the growth curve; On a solid aluminum plate containing 50mM, and Al added colony calmodulin inhibitor TFP and calcineurin inhibitor FK-506 compared to the smaller, growth was inhibited. Only added calmodulin kinase inhibitor KN-93 did not change significantly. Plate assay results with the results of the growth curve coincide.3. Western blot analysis of the impact the use of aluminum stress on and CNA CaM protein levels in C. humicola. The results show that with the increasing of aluminum concentration the expression of CaM protein gradually increased and the expression of CNA also showed a downward trend after the first rise. This change in transcription levels are substantially the same. These results indicate that aluminum stress can affect the level of transcription and translation levels of C. humicola CaM and CNA genes, CaM and CNA genes in C. humicola stress response rate process may play an important role.4. Effects of genetically modified yeast assay calmodulin and calcineurin in C. humicola aluminum tolerance. The results show that with the increasing of aluminum concentration flat panel detector, pYES3/CT-CNA and pYES3/CT-CaM two strains of genetically modified yeast resistance level of aluminum are significantly higher than the control yeast pYES3/CT. On a solid aluminum plate containing 0.1 mM compared with transgenic yeast, yeast pYES3/CT control the colony becomes apparent, indicating that growth inhibition. On a solid aluminum plate containing 2mM diluted 10"4, pYES3/CT-CNA and pYES3/CT-CaM two strains of genetically modified yeast can grow well, basically do not control the growth of yeast resistant aluminum. With increasing concentrations of aluminum, although transgenic yeast can grow, but gradually becomes smaller colonies, indicating that these two genes CNA and CaM promote growth, he ability to detect the content of the medium through the residual activity of aluminum, thus the detection of genetically modified yeast pYES3/CT-CNA and pYES3/CT-CaM aluminum absorption or adsorption. The results show that compared with the empty vector transfected pYES3/CT yeast, medium pYES3/CT-CaM pYES3/CT-CNA transgenic yeast and remaining active aluminum concentrations are significantly reduced. With increasing concentrations of aluminum, pYES3/CT-CNA transgenic yeast culture medium decreased the activity of the aluminum content is more obvious.The results show that CaM and its downstream target proteins CNA indeed involved in the response of C. humicola aluminum stress accordingly.In summary, calmodulin and calcineurin involved in the process of C. humicola resistant aluminum stress.