| Aluminum is one of the most abundant metal elements in the earth and also is the main factor which reduces crop yields in acid soils. However, it costs much and has no substantive change for aid deep soil that the way is to raise the pH of the soil through huge lime. It is one of the efficient ways for aluminum toxicity in acid soil to get stronger aluminum tolerance plants by genetic engineering. Therefore, the study is meaningful for agricultural production which is studying the mechanism of aluminum tolerance in biology and is finding the gene involved in the mechanism of aluminum tolerance.Microorganisms grow fast, short, easy to operate, is the study of the mechanism of resistance to aluminum better model organism. To study the mechanism of aluminum toxicity can provide a new perspective for the study in plants. The tea bush is a acid aluminum tolerance plant which have all kinds of aluminum resistant microorganism around the root. In this paper, a new yeast was isolated form the tea soil and identified as Cryptococcus podzolicus through the sequences analysis of D1/D2 region of 26S rDNA and ITS (Internal transcribed spacer, ITS) region. The strain is able to grow on the GM medium containing 200mM Al3+. In order to explore potential resistant-aluminum mechanism, the content of remaining activity aluminum decreased to 85.1% compared to the CK after cultivating for 24h through the ICP-AES, which may be adsorbed or absorbed by the yeast. The isolated strain provides a better experimental material for the study of the aluminum-tolerant gene and the aluminum-tolerant mechanism.As far as we know,14-3-3 protein is a highly conserved protein families in all higher eukaryotes, which is binding with over 200 kinds of protein involved in almost all biological processes in cells. Recently genome-wide studies on yeast strains with impaired 14-3-3 function supported the participation of 14-3-3 proteins in numerous yeast cellular processes. The gene sequence encoding the 14-3-3 protein was got through the early whole genome sequencing. In this paper, the recombinant 14-3-3 protein was purified and the 14-3-3 rabbit antibody was got through construction of prokaryotic expression vector for 14-3-3 gene. The content of 14-3-3 expression increase along with the increasing of the aluminum concentration and it is the largest at 20mM aluminum concentration through the Western Blot. The content of 14-3-3 expression and the content of plasma membrane H+-ATPase expression were the same with the change of the concentration of aluminum through the Co-immunoprecipitation between 14-3-3 protein and the plasma membrane H+-ATPase, which explained 14-3-3 protein and plasma membrane H+-ATPase were associated under aluminum stress.In order to verify the role of the 14-3-3 protein in Al-tolerant mechanism, the transgenic yeast for over-expressed 14-3-3 gene was got through the construction of 14-3-3 eukaryotic expression vector. The transgenic yeast could grow well in the culture medium which the concentration of aluminum was 5 while the PYES3/CT transgenic yeast and the negative control yeast could not grow or grow bad in the situation the the the concentration of yeast was diluted at a concentration of 10-4. Therefore, it explained over-expression of 14-3-3 protein enhances the tolerance of the yeast. Compared with the PYES3/CT transgenic yeast, the content of remaining activity aluminum in the PYES3/CT-14-3-3 transgenic yeast is lower with the increasing of increasing concentration of aluminum through the content of the residual activity aluminum. These data suggested that 14-3-3 protein plays a role in the resistance of aluminum. The results show that over-expression of 14-3-3 protein enhances the tolerance of the yeast, and the increasing of aluminum-tolerant in the transgenic 14-3-3 yeast may be due to the adsorption for the active aluminum. |
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