Cloning And Functional Analysis Of Phospholipase C1Gene In Fusarium Graminearum

Fusarium graminearum is an ascomycete that causes Fusarium head blight (FHB), which is a devastating disease of wheat, barley and other cereal crops worldwide. FHB epidemics occur frequently in central China and lower reaches of the Yangtze River especially. Cultivation system and global climate change have been implicated as a potential cause for increases in the severity and geographic distribution of FHB including Shandong province which belongs to northern China. F. graminearum is dominant species which can produce deoxynivalenol (DON) and derivatives acetyldeoxynivalenol (3-ADON or15-ADON), have a serious threat to human and animal health. However, the mechanism of toxigenic and pathogenesis of F. graminearum are not clear.Phospholipase C is known to be an important enzyme in the phospholipid signaling pathway, which can regulate a seroious of cellular processes including the biosynthesis and degradation of membrane lipids. Phospholipase C catalyzes the hydrolysis of phosphatidylinositol (4,5) bisphosphate [PtdIns (4,5) P2] to form Inositol (1,4,5)-trisphosphate P3(IP3) and diacylglycerol (DAG). IP3induce release of Ca2+from intracellular, and DAG, which can mediates the activation of calmodulin-mediated calcium-dependent proein kinase (PKC) directly. The typical modular structure of PLC consists of an N-terminal pleckstrin homology (PH) domain, a C-terminal C2lipid-binding domain, an EF-hand calcium-binding domain, and two conserved regions, the X and Y boxes.In this experiment, F. graminearum strain01-3and wheat cultivar yangmai158were available. F. graminearum PLCs were analyzed through the NCBI Blast, Conserved Domain Search GenBank and biology software DNAMAN on F. graminearum genome database; F. graminearum PLC1gene was cloned by gene specific primers; The gene knockout vector and completement vector was constructed.Finally we got the gene knockout and completement mutant by polyethylene glycol-mediated plastid transformation methods. Through analysis the change of FgPLCl knockout mutants and completement mutant on phenotype and pathogenicity, Find out the role of FgPLCl genes on sporulation and toxin synthesis of F. graminearum. The results were as follows:1. We have found five PLC genes on F. graminearum genome by NCBI Blast analysis totally.The relative location and structure of every PLC gene was defined. The PLCs were named as PLC1, PLC2, PLC3, PLC4, and PLC5. By NCBI Conserved Domain Search analysis, we found that the PLC1structure was similar to rice blast PLC1, both of them contain PH domin, EF hand domin, C2region, X and Y district. FgPLC1gene was cloned by gene specific primers from F. graminearum genome.2. The FgPLCl gene knockout vector pMDa+b+H was constructed based on the gene homologous combination theory and glycol-mediated-mediated gene transformation system. Transformants were screened by hygromycin B resistance and PCR with specific primers corresponding FgPLC1gene sequence and we acquired the gene knockout mutant AFgPLC1.3. The FgPLC1gene complementation vector was constructed based on the plasmid pCB1532. We transformed the complementation vector into AFgPLC1mutant, got the gene complementation transformation. It was confirmed that we got the gene complemented mutant After the PCR and RT-PCR analysis.4. Comparing the change of wild-type strain and mutant strains in growth, sporulation, gene expression, toxin synthesis, pathogenicity and other aspects, we have found that the fluffy aerial mycelium of FgPLCl knockout mutants was significantly reduced, Comapred with the wild-type strain, knockout mutant was inhibited on sodium chloride and Congo red medium,the inhibition rates were48.4%,28.2%;the inhibition rate of knockout mutant strain was4.4%on hydrogen peroxide medium, less than the CK. the growth of the wild-type strain was promoted but the knockout mutant was inhibited by6.64%on sorbitol medium compared with the PDA medium; the spores were short and thin,the quantity of spores doubled; the length decreased by82.5%, the width decreased by35.4%; The knockout mutant lost the ability to produce sexual spores; The gene expression level Tri5and Tri6have reduced by97%and76.5%on the knockout mutants respectively; otherwise, DON content also declined by76.5%. When replied the PLC1gene, these phenotype changes can be basically recovered. By these experiments, we can conclude that F. graminearum PLC1gene have a significant influence on mycelium growth, spore morphogenesis and toxin synthesis and other aspects.