Isolation And Purification Of Plant Activator Protein And Activity Detection

Plant activator protein is a new type of high-performance, multi-function, broad-spectrum and environment-friendly plant inducer isolated from Alternaria spp. JH505, which can induce plant to generate resistance to diseases and insects. A set of isolation and purification methods are reported in this paper, and we studied the activity of three major defence enzymes –POD, PPO and CAT, the dynamic variation of the level of H2O2 H and POD in tomato seedling after inoculating with Botrytis Cinerea,trying to reveal the mechanism of action of plant activator protein and provide theoretical basis for practical application, crystallization and biosynthesis of it. The results are presented as following: 1. Purification of Plant Activator Protein The purification program of 42KD plant activator protein was: 1. precipitating with 10-20% ammiaonia sulfate; 2. desalting with HitrapTM 26/10 column; 3. purifying by gel filtration chromatography with HitrapTM Q FF and SephacrylS-100 columns. After SDS-PAGE detection, a single band of 42KD was obtained. After detection and analysis, it shows that plant activator protein is an acidic protein with pI of 3.06. An amino acid sequence of N'end of plant activator was obtained. Through compare it with the data in GeneBank, no identical sequence was found, it could be concluded that plant activator protein is a new protein that haven't been reported. 2. Systemic induction of several defense enzymes in tomato leaves inoculated with Botrytis Cinerea and Plant activator protein was determined. After being treated with Botrytis Cinerea Persoo and plant activator protein, the activity of POD,PPO,CAT in tomato leaves were higher than the control. The results indicated that plant activator protein can induce the expression of these defense enzymes respectively in tomato leaves for POD,PPO and CAT,the optimum concentration of plant activator protein is 2μg·ml-1. and the induction of POD,CAT,H2O2 expression in susceptible tomato cultivar Fenguan was generally lower than in resistant tomato cultivar Zhongshu No. 6, The results showed that the induction of POD, PPO, CAT expression in tomato leaves was positively related to tomato resistance to fungal gray disease, and could be used as the indicators of tomato resistance to fungal gray disease. Tomato leaves treated with 2μg·ml-1 plant activator protein, which suppressed oxidize activation of hydrogen (CAT) in tomato leaves obviously, and increased the concentration of H2O2 . After treatment, CAT(1.85ΔA240·min-1·g-1) was 2.5 times compared with the negative control. the activation of CAT dropped to the negative control (treatment with sterilized water) level at 12h, then kept the same level with control. H2O2 concentration was increased gradually with the decreasing of CAT activation, and reached the highest level at 36h (134.67μmol·g-1) after treatment, increased by 30% compared with the negative control. The positive control treated with the salicylic acid was similar to above-mentioned trends. Tomato leaves treated with plant activator protein could also lead the change of POD isoenzyme strings concentration, and got the better systemic resistance to the Botrytis Cinerea, the control effect was up to 71.30%.Electrophoretic isozyme zymogram patterns of POD in tomato treated with Botrytis Cinerea and with Plant activator protein were studied. After inoculation with Botrytis Cinerea, the systematiec dynamic characteristics of POD isozyme was determined. The results demonstrated that plant activator protein can induce the expression of POD in tomato leaves, which were identified by the thickening of the original isozyme bands and the expression of some new isozyme bands. This thesis could not only help us to understand the mode of action of plant activator protein, but also provide information for the method of screening new protein biopesticides.