Study On Cloning And Expression Of Ascorbate Peroxidase And Glutathione Peroxidase Gene Of Salvia Miltiorrhiza Bge.

The generation and scavenging of Reactive Oxygen Species(ROS) is normally a dynamic balance in plant. Low levels of ROS could be as the signal molecular of stressment to participate in the physiological process, however, excessive ROS seriously damaged the proteins, membrane lipid, DNA and other cell compositions. Plants often encounter stress of bad environment during their growth and development, such as salt, drought, low temperature and so on. In which the salt stress seriously affects the growth of plants. ROS produce in plants when they are exposed to environmental stress. So, ROS must be scavenged in time. In order to reduce the oxidative damages, in the long run of evolution process, plants have formed a set of scavenging and protection machanism of ROS. They have two sorts of effective protection machanism of scavenge ROS, including enzymes promoting scavenge system and non enzymes promoting scavenge system. Enzyme-promoted-scavenging system mainly contain superoxide dismutase, ascorbate peroxidase, catalyse, glutathione peroxidase and so on. Non-enzymatic antioxidative molecules contain ascorbic acid, reduced glutathione and so on. The study on the genes of antioxidative enzymes is beneficial to the understand the physiology of plants under stress environment and is more useful to plants protection.In this research, seedlings of Salvia miltiorrhiza Bge. were used as experimental material. The SmAPX and SmGPX, which encoded ascorbate peroxidase and glutathione peroxidase distinctly, were cloned. Analyzing the characterization of nuclear acid sequences and the deduced amino acid sequences of the two genes, and predicting their secondary and spatial structures by bioinformatics methods. The tissue expression and expression under salt stress of the two genes were analyzed by fluorescence real-time PCR. And also the expression vectors of SmAPX and SmGPX in E.coli M15 were constructed to analyze their expressions. The main experiment methods and conclusions of the thesis are as follows:1.Based on the sequences of Salvia miltiorrhiza Bge. EST database, we cloned a gene named SmAPX(758bp) by RT-PCR, which contained an ORF of 753bp and encode 250 amino acids. The nucleic acid and the deduced amino acid sequences were analyzed by bioinformatics tools and concluded that it was a cytosolic protein which had no trans-membrane domains. The SmAPX expressed in roots, stems and leaves, but lowest in roots, higher in stems and highest in leaves, 20 folds higher in leaves than in roots. The expression increased with the prolonged stress time and increasing salt concentration. The expression of SmAPX was two folds higher when he concentration of NaCI was 200 mM and materials were exposed to salt stress for 72 h, and highest when the concentration of NaCl was 200 mM and materials were exposed to salt stress for 48 h, which increased seven folds compared to control. The expression decreased when the concentration was 300 mM, but also higher than control. A protein of about 27 kD was induced by transforming the SmAPX to E.coli M15, which increased the salt tolerance of E.coli M15 in some way.2. Based on the sequences of Salvia miltiorrhiza Bge. EST database, we cloned a gene named SmGPX(758bp) by RT-PCR, which containe an ORF of 501bp and encoded 166 amino acids. The nucleic acid and the deduced amino acid sequence were analyzed by bioinformatics tools and concluded that it was a cytosolic protein which had no trans-membrane domains and was possiblely phosphorylated at 12 positions after translation. It may be a putitive phospholipid hydroperoxide glutathione peroxidase which belonged to a member of GPXs and protected biomembrane from oxidative damages according to the characteristic sequences of PHGPX. The SmGPX was also cloned from DNA level and existed in genome of Salvia miltiorrhiza Bge. with low copies by Southern blotting analysis. The SmGPX expressed in roots, stems and leaves, but lowest in roots, higher in leaves and highest in stems. The expression ins 6.78 times of it in roots. The expression increased with the prolonged stress time under 250 mM NaCl stress. A protein of about two-folded large in molecular mass was induced from Ecoil. M15.