The Cloning And Preliminary Functional Studies Of Alternative Oxidase (AOX2) In Cerasus Humilis (Bge.) Sok

Drought stress, one of the most common natural disasters, results in a series of problems, such as, decreasing of crop production to a large extent and desertification of land. Cultivating new-type drought-tolerant plants is an effective approach to solve these problems. Cerasus humilis (Bge.) Sok. has very strong ability for drought resistance and has very high economic value. All these make C. humilis a popular species for growing on dry land. To adapt to harmful conditions, plants under drought stress regulate gene expressions through signal transduction mediated by ABA. In this process, alternative oxidase (AOX) plays an important role. To explore the relationship between stress and C. humilis plants, we assayed the expressions of AOX and uncoupling protein (UCP) families under the treatment of drought stress and salicylic acid, respectively. Then, two prokaryotic expression vectors and two plant expression vectors were constructed successfully by using molecular biology methods. The results are as follows:1. We found that the expression levels for almost all members of the AOX and UCP families increased under drought stress, except that AOX1a and AOX2 changed rarely. Among them, expression of AOX2 showed the highest level in the whole period of treatment. In contrast, all members of the two families expressed a lower level except UCP2 after treatment of salicylic acid. However, the expression of AOX2 showed the highest level all the same, speculating that AOX2 is a key gene for C. humilis in response to stress.2. Full length of AOX2 gene in C. humilis contains 1559 base pair. It’s 5’noncoding region and 3’noncoding region contains 185 and 345 base pair, respectively. It’s open reading frame contains 1029 base pair, coding 342 amino acids. Many alpha helix and coil were found in AOX2 of C. humilis. Two transmembrane domains are included. It’s conserved domains focus on the side of C-terminal. The homology of AOX2 from C. humilis and Arabidopsis thaliana is 66.96%,1.98% higher than the average of the 22 species.3. We found that pGEX-4T-3-A can not be stably expressed in BL21(DE3) and DH5a, except in Rosetta(DE3). The amount of dissoluble fusion protein is immensely less than the fusion protein in precipitation. The optimizing conditions for inducing is 0.2mmol/L IPTG,15 ℃,180rpm,30h. After purification, a specific protein band can be gained, which has been identified as GST-AOX2 by Western blot analysis. According the final dentified results, we successfully transformed AOX2 of C. humilis into Arabidopsis thaliana, and obtained the inheritable T2 transgenic plants.