Team:GZHS-United/Design
Design
Experiment A
For experiment A, as we all know, terrestrial plants always suffer from a higher stress under the strong light and high temperature than the marine plants. Also, from the essays we read, we know that Arabidopsis —— a kind of higher plants, has a high enzymatic activity of the APX.
So we suppose that if the APX of Arabidopsis has a higher stress resistance under the exposure to high temperature and strong light compare to the APX of zooxanthellae. During this experiment, we amplified the APX gene sequence from the cDNA of Arabidopsis by PCR. Then, we linked this sequence to pET28b-rplJ plasmid by the enzyme and transferred into competent cell BL21 by means of heat shock transformation to grow. After the growth of bacterial strains, we obtained the cell effluent liquid by ultrasonic fragmentation, purified it by His-Tag Protein Purification, and measured and compared their APX activity—— AsA oxidation rate to wide type using APX activity detection kit and getting the results by enzyme activity calculation formula. If the enzyme activity data conform to the prediction, the activity difference is large, it indicates that the prediction above is correct and can be used for further exploration.
In addition, in order to find the difference of tolerance between zooxanthella APX(SymAPX3) and Arabidopsis APX(ATAPX1) under various condition of temperature, illumination and pH, we further design experiment B to obtain more sufficient data to analyze the potential of ATAPX1, and treating the protein samples respectively in different temperature, illumination and pH gradients and measuring the activity of sample protein enzyme.
Experiment B
For experiment B, from a new perspective, we are confusing that if we can increase the APX enzyme activity and its stress resistance just in zooxanthella itself. conjecture of hydrogen peroxide binding site in the APX gene of zooxanthella: amino acids with electric charge in the APX gene of zooxanthella have certain influence on the rate of hydrogen peroxide decomposition by APX. So we try to verify this hypothesis. First, we selected the APX gene of zooxanthella and named it SymAPX3 from the websites KEGG and NCBI.
The two new sequences were the first SymAPX3-mut1, which mutated R and L directly to A, and the second SymAPX3-mut2, which mutated all charged amino acids —— R, H, D to A. and edited them into pET28b plasmid. We turned these edited sequences over to biological companies for synthesis, and compared its APX activity with that of zooxanthella wild type by the same procedures as above.