| In our previous study, it was found that there were four MAPK(Mitogen-Activated Protein Kinase) cascades in Setosphaeria turcica by bioinformatics methods, in which HOG-MAPK cascade was involved in hyperosmotic stress. Additional, St FPS1 gene of Setosphaeria turcica, an aquaglyceroporin gene, was cloned and the function of the gene had been preliminarily identified by RNAi and knock-out technique. In this study, the function of St FPS1 will be proved by gene knock-out technology again. The function of St FPS1 will be confirmed by comparing the growth, development and response to hyperosmotic stress of the mutants with those of WT(the wild-type strain). The main findings were as follows:1. Two St FPS1 knockout mutants were obtained in the experiment. St FPS1 knockout vector of S. turcica, which was constructed in previous study, was verified firstly and then was transformed to the protoplasts of S. turcica. Two St FPS1 knockout mutants were identified by hygromycin B resistance screening, special primer PCR and RT-PCR.2. St FPS1 affects the morphogenesis of the mycelium cells and the yield of conidia. The growth rate of St FPS1 gene knock-out mutants was inhibited. Hyphal cells enlarged and some of them even turned swollen compared with WT. The mutants failed to produce conidia any more. The results showed that St FPS1 gene not only affected the mycelium cell development but also affected the production of conidia.3. St FPS1 is involved in osmotic stress reaction of mycelium cells. Hyphal cells of St FPS1 mutants turned swollen and significantly increased the glycerol content compared to WT. The inhibition ratio of the mutants under osmotic stress was much lower than that of WT. The results showed that St FPS1 affected hypertonic stress response and acted as a negative regulatory factor in the pathogen. In addition, the expression levels of key genes of glycerol synthesis(St GPD1, St HOR2) in the mutants elevated under hyperosmotic stress but were still under the value of WT under common culture condition. Based on the results above, we conferred that the main reason of increased significantly glycerol content in the cells of the mutants under osmotic stress was the lack of the aquaglyceroporin Stfps1 and glycerol efflux was blocked.4. St FPS1 was associated with the development of the cell wall of the pathogen. The cell wall of the mutant turned significantly thinner than that of WT under transmission electron microscopy. Compared with WT, the cell wall of St FPS1 gene knock-out mutants were the expression of the cell wall synthesis-related gene, such as St HOG1 and St SLT2 was detected using real-time RCR technique, the expression level of St HOG1、St SLT2 decreased significantly; the mutants became less sensitive to cell wall synthesis inhibitors. Real-time RCR showed that the expression level of genes, which were related to the synthesis of cell wall, such as St CHS1、St CHS2、St CHS2、St Rho1, lowered. Moreover, the sensitivity to the interferents in the process of cell wall biosynthesis in the mutants cut down. The results indicated that St FPS1 was related to the morphogenesis of the cell wall of S. turcica.5. Under osmotic stress, the thickness of cell wall in WT and the mutants are increased, but mutants significantly thicker. Also, the genes related to cell wall synthesis, such as St CHS1, St CHS2, St CHS3, and St Rho1, increased their expression level in the condition, suggesting that increasing cell wall thickness was one of the important reaction for the pathogen under osmotic stress.6. St HOG1, a key gene in HOG-MAPK pathway and St SLT2, in CWI-MAPK pathway, decreased their expression level in St FPS1 knockout mutants. Thus, the expression level of cell wall synthesis enzyme genes lowered and they blocked the synthesis of cell wall. The results suggested that St FPS1 was associated with HOG-MAPK and CWI-MAPK cascade pathway.7. St FPS1 was involved in oxygen stress and temperature stress reaction in S. turcica. |
PDF Full Text Request