| Downy mildew is an important disease of cucurbits caused by the obligate pathogen Pseudoperonospora cubensis. It has led to substantial economic losses to cucumber growers. Plant chitinase is an important group of pathogenesis-related proteins and has the activity of degrading chitin in the walls of fungi to inhibit fungal infection. Plant chitinase mainly distributes in intercellular space and vacuole. Chitinase in plant intercellular fluids is frequently induced in the first place by pathogen infection. As a result, the study on the change of induced chitinase in intercellular fluids is a significant part of revealing the mechanism of plant defense responses.1. In this study, after inoculating with downy mildew, the dynamic changes of induced chitinase in intercellular fluids of cucumber leaves and the seedling growth of cucumber with different resistance were studied. As shown by the results, after the inoculation, the content of soluble protein of the resistant cucumber variety (Cucumis sativus cv. Jinchun No.4) was faster and higher than that in the susceptible variety (cv. Changchunmici). The activity of chitinase in the resistant variety had a rapid change, leading a peak at the second day, and maintained at high levels until 7 days; the evident expression of a 27kDa chitinase was found at the second day by SDS-PAGE method, and had showed visible accumulation at the fourth and seventh day. This was further confirmed by western blotting. At the same time, ethylene production of the resistant cucumber leaves grew up rapidly after the inoculation, while the susceptible variety had a large range of increment at the fourth and seventh day. In addition, the fresh weight and accumulation of dry matter of the susceptible cucumber variety were decreased at the fourth and seventh day after the inoculation. But those indexes of the resistant variety were much more than the susceptible at the seventh day. The above results indicated that the relationship between induced chitinase in intercellular fluids and cucumber resistant potential to downy mildew were closely related. 2. Ethylene is considered as a kind of messenger molecule in plant defense mechanism to against pathogen attack and has a close relationship with pathogenesis-related protein formation in plant defense responses. Ethephon is an artificial compound. It can be easily absorbed by plant and then release ethylene. In this experiment, after spraying with different concentration of ethephon, the dynamic changes of induced chitinase in intercellular fluids of Jinchun No.4 cucumber leaves and cucumber seedling growth were studied. As shown by the results, after the treatment, the content of soluble protein was higher than the check group. The activity of induced chitinase had a different range of increasing by using different concentration of ethephon, and the 0.02% ethephon was the most effective. The evident expression of a 27kDa chitinase was found in 24h after 0.02% ethephon treatment by SDS-PAGE method, and had showed visible expressions until 7 days. That was further confirmed by western blotting. In addition, after the 0.01% and 0.02% ethephon treatment, the increment of height growth of cucumber seedlings was much more decreased than the check group;the stem diameter growth was increased significantly by 0.02% ethephon treatment. The above results indicated that ethephon could induce chitinase in intercellular fluids of cucumber leaves. At the certain range of ethephon concentration, the induced effect of the higher concentration was much more efficient. And it had showed that the formation of induced chitinase had a close relationship with cucumber sensibility to ethylene concentration.This study shows that the 27kDa chitinase in intercellular fluids of cucumber leaves could be induced by inoculating with Pseudoperonospora cubensis or ethephon treatment. And it is indicated that the relationship between induced 27kDa chitinase in intercellular fluids by inoculating and ethylene production of cucumber leaves are closely related. |
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