The Purification Of Chitinase From Alternanthera Philoxeroids And The Study Of Its Response To Some Stress

Because of its strong resistance to lots of stress factors, Alternathera philoxeroides, an alien invasive plant, has become a harmful weed in China rapidly. It polluted the environment and influenced the development of agriculture and fishing severely. To prevent it from the spreading has been the main task of the governments all over the worlds. But the control of it by chemical and biological method was not perfect.Pathogenesis-related proteins (PRs) are functional proteins in plant induced by pathogens and/or environmental stresses to increase the resistant ability of plant. Chitinase is one of the PRs related to stress reaction in many plants.So, in order to find the proper moment for chemical and biological control of Alternathera philoxeroides or to obtain other way to control this plant, we intended to explore its resistance mechanism, to observe the change of its chitinase activity when exposed to different stresses, to purify and assay the enzymatic properties of its chitinase. The results were followed:Firstly, the chitinase of Alternathera philoxeroides was induced enzyme. Secondly, the Alternathera philoxeroides responded to 4 stress factors in different ways.It seemed that this plant was very sensitive to ethylene, because its chitinase activity increased significantly as soon as it was treated by ethylene( p<0.01 ), and still had higher enzyme activity in 2th and 3 rd days (p<0.01 ); When exposed to chitosan, Alternathera philoxeroides produced more chitinase 2-4 days after treatment ( p<0.05 ); And injury also could increase the amount of chitinase in its leaves 3 days after cut. But, compared to these three factors, salt had lighter effect on this plant, it caused the increment of chitinase of Alternathera philoxeroides only 8 days after the plant cultured with 1% NaCl( p<0.01 ).Lastly,the chitinase of Alternathera philoxeroides had its special properties.The chitinase was extracted from the leaves of Alternathera philoxeroides treated by ethylene, precipitated with 40%～60%ammonium sulfate and purified by Sephadex G-75chromatography, DEAE-Sepharose Fast Flow and Sephadex G-100 chromatography. The purified enzyme showed a molecular weight about 45KDa proven by sodium dodecylsulfate polyacylamide gel electrophoresis (SDS-PAGE). Its Km value for chitin was 0.58 mg·ml﹣1. It was stable in the range of pH 4.0 to 8.0 and almost had no activity at pH 2.0 or 10.0. Its optimum pH was about 5.7. This enzyme also could endure high temperature and began to loss its activity when temperature was higher than 80℃. Its optimum temperature was about 60℃.It was suggested that the chitinase played an important role in the response of Alternathera philoxeroides to ethylene, chitosan and injury stress. And maybe the tolerance of this enzyme to high temperature was related to the strong resistance of Alternathera philoxeroides.