Fuction Analysis Of Glucosylceramide Synthase In Colletotrichum Gloeosporioides Of Postharvest Fruit

The pathogenic fungi of postharvest fruit have caused great loss to the postharvest,and the pathogenic bacteria have begun to resist the commonly used azole and multi thinning fungicides.Therefore,the research and development of the new fungicides are very important for the sustainable development of the fruit industry.Sphingomyelin is not only an important component of the plasma membrane,but also a signal molecule involved in the regulation of many kinds of life activities.In animal pathogenic fungi,it has been proved that sphingolipids play an important role in mycelium formation,spore germination and pathogenicity,and the structure of sphingolipid synthesis related enzymes is significantly different from that in animals.Therefore,the mycelial sphingolipid metabolic pathway has become a candidate target site for the screening of new type of antiviral drugs in animals.In this study,an important fruit postharvest pathogenic fungus,Colletotrichum gloeosporioides(C.g),which was used as the material.Based on the sequence of sequencing,the key gene of sphingolipid synthesis,glucosylceramide synthase(GCS),which was isolated and cloned from the strain of the bacteria through the GCS gene.The effects on the growth,appressorium formation,sporulation and pathogenicity of the Colletotrichum gloeosporioides by knocking and complementing the GCS gene.The effect of exogenous GCS ds RNA on the pathogenicity of Colletotrichum gloeosporioides were further analyzed.We hope to clarify the specific functions of GCS gene of Colletotrichum gloeosporioides.To provide theoretical support for further prevention and control of pathogens.And the specific results are as follows:1.Acquisition of deletion mutants and complemention mutantsBy using the Gateway gene homologous recombination technology,six gene deletion mutants were obtained by using the hygromycin B(Hyg B)resistance gene fragment to replace the GCS gene ORF in the C.g genome,named ?Cg GCS1,?Cg GCS7,?Cg GCS10,?Cg GCS11,?Cg GCS14,and?Cg GCS22?The correct substitution in genome was preliminarily verified by PCR.The deletion mutants showed resistance to Hyg B and stable inheritance.A similar method was used to integrate the genomic sequence of the GCS gene(including the promoter and the terminator)into the mutant by using the Cg GCS1 as the material,and five genes were obtained,named ?CPCg GCS1,?CPCg GCS2,?CPCg GCS3,?CPCg GCS4 and ?CPCg GCS5?Quantitative PCR analysis showed that GCS gene expression was almost not detected in the mutant,while the expression of GCS gene in the complement mutant showed a similar level to that in the wild type.This provides a good mutants for further study of the function of Cg GCS gene.2.The effect of GCS gene mutants on mycelial growth,spore germination,appressorium formation,sporulation and pathogenicity of the pathogen.Compared with the wild type,the mutants growth rate of the mycelium decreased obviously,and mycelium became twisted and the branch increased.The rate of spore germination and appressorium formation were significantly lower than wild type.Moreover,under normal conditions,the mutants hardly spores.In addition,the infection rate of the Cg GCS mutants on tomato and mango fruits slowed down significantly,also the pathogenicity decreased by more than half compared with wild type strain.The phenotypes of complement mutant strains were consistent with wild type strain.The above results indicate that Cg GCS gene plays an important role in mycelial growth,spore germination,appressorium formation,sporulation and pathogenicity of pathogens.3.The effect on the synthesis of sphingomyelin by deleting Cg GCS gene.The sphingolipid substances in the pathogen were analyzed by HPLC-MS/MS technology.The results showed that the deletion of Cg GCS resulted in obvious changes in the sphingolipid substances.Compared with the wild type strain,the total content of LCB and g Cer decreased significantly in the deletion mutant and the total h Cer content increased which were 0.26 times,0.03 times and 4 times of the wild type respectively.Specifically,although the total LCB content was decreased,the d18:0 content in the 5LCB detected increased.GCS upstream substrate h Cer content increased,especially the detection of d19:2 h18:0 and d18:2 h18:0 increased by 11.6 times and 5.25 times respectively.For g Cer,the content of the five detected g Cer decreased sharply,especially the d19:2 g18:0 and d19:2 g18:1,which were only 0.2719% and 5.48726% respectively of wild type.The above results show that GCS gene we identified is a true glucosylceramide synthetase gene.On the other hand,the GCS gene deletion significantly affects the metabolic process of the sphingolipid.4.Comparison and analysis of mutant and wild type transcriptional groupTranscriptome analysis showed that 264 genes were upregulated while 317 genes downregulated compared with wild type.GO analysis showed that the differentially expressed genes were mainly involved in metabolic related biological processes,and their expression products mainly distributed in the membrane related parts to catalyze and bind related activities.KEGG analysis showed that the differentially expressed genes were mainly concentrated in metabolic related biological pathways.Further analysis showed that an important pathogenic factor of Colletotrichum gloeosporioides,the expression of pectate lyase(pectate lyase)gene which decreased significantly in the mutant,indicating that glucosanceramide could regulate the pathogenicity of the pathogenic gene by affecting the expression of pathogenic genes.5.Effect of Cg GCS gene ds RNA in vitro inoculation on pathogenicity of Colletotrichum gloeosporioidesThe ds RNA of the Cg GCS gene fragment was transcribed in vitro,and then ds RNA was inoculated in the tomato fruit before the pathogen was.It was found that the ds RNA of the Cg GCS gene fragment could obviously decrease the infection of the pathogen,and the pathogenicity decreased by more than half.