| Phosphoinositide-specific phospholipase C (PI-PLC) is a critical enzyme in thephospholipid signaling pathway. Signals outside the cell wall are perceived at the cellmembrane by receptors linked to a variety of signaling pathways, especially phospholipidsignaling pathway. In this pathway, signals activate PI-PLC, which catalyzes the hydrolysis ofphosphatidylinositol (4,5) bisphosphate [PtdIns (4,5) P2] to form diacylglycerol (DAG) andInositol (1,4,5)-trisphosphate P3(IP3).IP3triggers the release of Ca2+from intracellularstores, and DAG with Ca2+mediates the activation of calmodulin-mediated calcium-dependentprotein kinase (PKC) directly. As intracellular second-messenger molecules, they convert andtransmit the external signal, triggering a series of intracellular reactions.Cucumber is an important commercial vegetable. With the cucumber cultivation area hasexpanded each year and crop-culture intensification and specialization, diseases is veryprominent during cucumber production. The cucumber fusarium wilt and other soil-bornediseases is becoming increasingly serious, affecting the yield and quality seriously. In thisstudy, with Fusarium oxysporium and cucumber seed as experimental materials, monosporousstrains of Fusarium oxysporium were separated. We analyzed PLCs in Fusarium oxysporium f.sp. Lycopersici genome database by Blast, Conserved Domain Search and DNAMAN andcloned the complete sequence of FoPLC4. The FoPLC4gene replacement vector andcomplementation vector were constructed. The role of FoPLC4gene in the pathogenicprocess was explored by analyzing the phenotype and pathogenicity of mutants. The resultsare as follows:1. According to Koch’s postulates, the identification of virulence of isolated strains showedthat Fusarium oxysporium was the pathogen of cucumber wilt disease. Select some strongestvirulence strain from45monosporous strains.2. Through BLAST, Conserved Domain Search and DNAMAN We found that Fusariumoxysporium f. sp. Lycopersici has nine PLC genes, which have the typical structural featuresof PLC. We named them according to their positions on the chromosomes in the genome.Analysis of the amino acid sequences of PLCs revealed that the domain structures areidentical between PLC4and PLC6-they have the four basic structures of the PLC gene, whilethe others do not. PLC1and PLC9have not EF domain however they have PH domain andC2domain and catalytic domain. PLC2，PLC3，PLC7and PLC8only have catalytic domainand C2domain. PLC5has no PH domain. PLC1, PLC6and PLC9have the higher gomology. 3. Primers were designed according to the PLC4gene. We cloned the complete sequence ofFoPLC4. Sequence analysis of the FoPLC4gene indicated that it included a3282bp DNAwith no intron and its predicted protein contained1092aa. The231-334amino acida is PHdomain. The419-504amino acida is EF domain. The510-653amino acida is X domain. The775-859amino acida is Y domain. And the893-1041amino acida is C2domain.4. The FoPLC4gene replacement vector was constructed based on the gene homologouscombination theory and PEG-mediated gene transformation system. Transformants werescreened by hygromycin B resistance and PCR with specific primers corresponding tohygromycin phosphotransferase or FoPLC4gene and confirmed by RT-PCR analysis.5. The FoPLC4gene complementation vector was constructed based on the plasmidpCB1532. After transforming the vector into FoPLC4mutant, the sulfonylurea(SUR)-resistant transformants were screened by PCR and confirmed by RT-PCR analysis.6. Although the wild-type and mutants of FoPLC4gene were similar in vegetative growthrate, the mutant FoPLC4exhibited sparse, fluffy aerial mycelium, significantly reducedsporulation, and longer conidia. The sporulation reduced82.2%compared with the wild-type,and disease index was approximately decrease by53.3%. Its pathogenicity was weakenedsignificantly. The results showed that FoPLC4gene regulated the hypal growth, sporulation,morphogenesis of conidia and pathogenicity. |
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