Cloning Of Four APX Gene And Promter And Analysis Of Catalytic Properties In Populus Tomentosa

The environmental stress results in production and accumulation of reactive oxygen species (ROS) in plant cells. ROS can cause extensive cell injury if they can not be scavenged efficiently. APX which is a protein that contain hemoglobin is an important enzyme of enzymatic detoxication system in plant and plays an important role in scavenging H2O2 and in protecting cells against the toxic effects of H2O2 in higher plant. In this paper, we cloned promoter sequences of three APX genes(homologous gene located on chromosome LG-â…¡,LG-â…¤,LG-â…¨). We accomplished the prokaryotic expression of four APX genes (homologous gene located on chromosome LG-â…¡-Scaffold-57,LG-â…¤). We analyzed the enzamatic characteristic of two APX genes (homologous gene located on chromosome LG-â…¡,Scaffold-57) and the expression of APX gene (homologous gene located on chromosome Scaffold-57) in different tissues of 7-8 years old Populus tomentosa. Following showed the main research and the result of experiment:1. Cloning of APX gene promoter sequence:By the method of electrical cloning, combined with the genome sequence of Populus trichocarpa, we inferred APX gene promoter sequence in Populus trichocarpa. According to the highly homologue between Populus trichocarpa and Populus tomentosa, we referred APX gene promoter sequence in Populus trichocarpa to design the primers.Then using DNA of Populus tomentosa as template, we cloned three APX genen promoter sequence in Populus tomentosa(length:950bp,1405bp,1234bp).2. Enzymatic characteristic analysisi of APX gene:Using 1mM IPTG inducing Pet30a-(+)-PpAPX prokaryotic expression vector. In this paper, we purified two APX gene protein (homologous gene located on chromosome LG-â…¡,Scaffold-57) by Ni2+-NTA affinity chromatography and analyzed the enzymatic properties of these two proteins. During the process of prokaryotic expression, APX gene(homologous gene both located on chromosome LG-V) formed a large number of indusion bodies. In this paper, we utilized urea washing,dissolving indusion bodies and refolding protein. Afterwards we purified the protein by Ni2+-NTA affinity chromatography to obtain higher purity recombinant protein.3. Real-time quantitative analysis of APX gene:Using real-time fluorescence quantitative PCR, the mRNA levels were quantified for one APX gene(homologous gene located on chromosome Scaffold-57) in different tissues of Populus tomentosa of 7-8 years. We found that the expression of the APX gene in mesophyll of old leaf is the most, followed by the expression in vein of the old leaf, and the least expression in cambium.