Strcture And Function Analysis Of StFPS1Gene Encoding An Aquaglyceroporin In Setosphaeria Turcica

In our previous study, it was found that there were three MAPK (Mitogen-ActivatedProtein Kinase) cascades in Setosphaeria turcica, in which HOG-MAPK (High-OsmolarityGlycerol-MAPK) cascade was involved in hyperosmotic stress. An aquaglyceroporin genenamed as StFPS1, located in the downstream of HOG-MAPK cascade and was probablyinvolved in hyperosmotic stress in S. turcica. In this study, StFPS1gene sequence will besearched from the genome database of S. turcica and StFPS1gene knock-out mutant willbe created by gene knock-out technology. Furthermore, the function of StFPS1gene willbe identified by comparing the differences between the mutant and the wild-type strain.The mainly results were as follows:1) Two kinds of aquaglyceroporin, FPS1-like and Yfl054-like, were found by searchingthe genome database of S. turcica published on JGI website(http://genome.jgi-psf.org/).2) A1393-bp gene named StFPS1was cloned in S. turcica. The gene contained a1393-bp DNA sequence, an1179-bp ORF (open reading frame),4introns and5exons.3) Based on the basic plasmid pBS-pUC, a gene knock-out vector of StFPS1wasconstructed. The recombinated vector was transformed to the protoplasts of S. turcicamediated by PEG3350.27transformants were obtained by culturing on PDA mediumcontaining50μg/mL hygromycin B. At last, one mutant was determined throughspecial prmer PCR, RT-PCR and Southern blotting.4) StFPS1gene knock-out mutant showed lower colony growth rate, abnormal myceliumdevelopment, no conidia, indicating that StFPS1gene was involved in regulating thedevelopment of mycelium and conidium.5) StFPS1gene knock-out mutant exhibited thin cell wall and was sensitive to cell wallsynthesis inhibitors, suggestting that StFPS1gene regulating the cell wall developmentand its integrity.6) StFPS1gene knock-out mutant emerged increased growth rate, swollen hyphal cells,threefold glycerol content higher than wild-type strain, revealing that StFPS1gene wasa negative regulatory factor in hyperosmotic stress reaction.7) The virulence and component of crude HT-toxin produced by StFPS1gene knock-out mutant had no difference with that of the wild-type strain. The activities of cell wallcatabolic enzymes and the infection abilities of the mutant did not changed. The resultsindicated that StFPS1gene was not involved in regulating the produce of HT-toxin andpathogenic enzymes activity.