Prokaryotic Expression And Antibody Preparation Of Populus Euphratica CPD Gene And Its Expression In Transgenic Arabidopsis

Brassinosteroid (BR), a recently discovered plant hormone, plays a very important role in the growth and development of plants. Most of the existing studies on BR biosynthesis have been done on annual herbaceous plants, such as Arabidopsis thaliana and rice, with few on the perennial woody plants. CPD (constitutive photomorphogenesis and dwarfism) is related with BR biosynthese. Recently, a full-length of cDNA sequence homologous to Arabidopsis CPD has been cloned from Populus euphratica, a tree species with extremely high tolerance to drought, high temperature and alkali, named PeCPD, at our lab. For futrher understanding the subcellular location of the protein encoded by PeCPD, a prokaryotic expression vector for PeCPD was constructed, the vector was successfully induced to express in bacterial, the expressed PeCPD protein was purified and corresponding antibody was prepared, in this study. Finally, the antibody was used to detect the expression profile of PeCPD gene in various Arabidopsis-PeCPD transgenic lines. The main results of my experiment are listed as follows:1. The pET30a-PeCPD prokaryotic expression vector was constructed and was induced to express highly in E.coli Rosetta (DE3). SDS-PAGE detection showed that the expressed target protein was presented mainly in the form of inclusion body. LC-MS/MS identification confirmed that the presumed target protein was just the product of PeCPD gene;2. The PeCPD protein was purified by the methods of Ni2+chelate affinity chromatography (Ni2+-CAC) and the differential centrifugation (DC) followed by preparative SDS-PAGE, named DC-PSDS-PAGE. The purity and yield of the obtained target protein of the two methods are0.816mg/L and30%for Ni2+-CAC and37.897mg/L and95%for DC-PSDS-PAGE, the latter being apparently better than the former;3. The purified PeCPD protein was used as antigen to raise corresponding antibody by immuning rabbits. ELISA detection suggested the antibody titer was1:320000. The double-diffusion assays in agarose gel experiment showed a single line and a1:3sensitivity. Dot blot analysis showed that its minimum sensitivity was1:3000. These results showed that the antibody met the requirement of the subsequent experiments;4The PeCPD antibody mentioned above and western blot method were employed to detect the expression of PeCPD in Arabidopsis thaliana wild type (Atwt). cpd mutant (Atcpd), Atcpd-PeCPD transgenic lines and Atwt-PeCPD transgenic lines. The result showed that there were some non-target hybrid bands except the target band. So the antibody specificity is not satisfied and further studies are required.