Cloning And Functional Analysis Of ?-1,6-glucanase Gene In Fusarium Oxysporum F.sp.cubense Race 4

The banana fusarium wilt is caused by Fusarium oxysporum f.sp.cubense?Foc?,is one of the most devastating disease of banana.Based on the pathogenicity of Foc,it can be divided into four races.Among of them,the race 4?Foc4?can infect almost all banana varieties.Cellulase as one of pathogenicity factor,play an important role in the infection process of Pathogenic fungi that helps degrade host cell walls.The Foc4 secretion proteome has been studied in our laboratory and it was found that the expression of?-1,6-glucanase in Foc4 was significantly up-regulated after induction of banana tissue,it was speculated that it may be related to the pathogenicity of Foc4,but there is no report on the function of?-1,6-glucanase gene in Foc4 yet.The function of?-1,6-glucanase gene in Foc4 was studied for the first time by gene cloning,eukaryotic expression and gene knockout methods.The specific results are as follows:?1?The real-time fluorescent quantitative PCR technique was used to verify the change of the expression of Foeg1 in Foc4.The results showed that the expression of Foeg1 in transcriptional level was significantly up-regulated in Foc4 under the condition of banana tissue induction,which was consistent with the results of secreted proteome.?2?The?-1,6-glucanase gene in Foc4 was cloned and sequenced,and a DNA sequence of 1381 bp and a 1233 bp cDNA sequence were successfully obtained.The molecular weight of Foeg1 encoded protein is 46923.26,isoelectric point?pI?is 8.27,total number of atoms is 6504,molecular formula is C₂₁₃₃H₃₁₈₆N₅₇₂O₅₉₆S₁₇,and theoretical instability index is 28.02.The?-1,6-glucanase gene of Foc1 was cloned and sequenced by the same method,and the sequences of the?-1,6-glucanase genes of the two races were compared and found.There is only one base difference in the DNA sequence and the cDNA sequence is exactly the same.?3?The recombinant eukaryotic expression vector for Foeg1 in Foc4 was successfully constructed.Methanol was used to induce expression,and a target band that matched the expected size was obtained.The cellulase activity of the induced product was further examined.The results showed that the protein induced for 48 h could leave a yellowish hydrolytic circle on the Congo red plate and had cellulase activity.?4?The?-1,6-glucanase knockout mutant of Foc4 was obtained by ATMT-mediated gene knockout.The phenotypic analysis showed that the growth rate of Foc4-?Foeg1 on PDA plates was significantly lower than the wild-type,and the colony edges were not neat,hyphae were not fluffy,and purple-red pigment began to appear on the third day of culture.The growth rate of Foc4-?Foeg1 on SM medium containing 1%CMC-Na was also significantly lower than the wild type,indicating a decrease in the utilization of cellulose.The results of sporulation statistics showed that the sporulation of?Foeg1 was significantly less than the wild type,but there was no significant difference in spore germination.The results of pathogenicity analysis showed that there was no significant difference in the virulence between the wild-type and?Foeg1.