Functional Analysis Of Heat Shock Protein On Verticillium Lecanii Against High Temperature Stress

Verticilium lecanii is a kind of entomopathogenic fungi which can be used to control plant diseases and insect pests.The stability of the storage process and the insecticidal effect of biocontrol fungi is an important reason for its application and popularization.Temperature is an important factor affecting the storage and field infectivity of fungal spores.Heat shock proteins play an important role in improving the heat resistance of organisms.Therefore,it is important to study the heat resistance mechanism of heat shock proteins to entomopathogenic fungi in order to solve the problems of the development and application of fungi.In this study,VL17 and VL18 strains were tested and compared,and the effects of temperature on aphid killing activity,mycelial growth and spore germination of two strains were compared,and then to compare the difference of heat shock protein gene expression under heat stress to find out the key heat shock proteins,which play a major role in the heat stress,cloning the key heat shock protein genes and constructing the key heat shock protein gene knockout strain to biological characteristics of the original strains and knockout strains.In this study,the function of heat shock protein in the resistance to high temperature stress in Verticillium lecanii was revealed,which lay a theoretical foundation for the popularization and application of the preparation.The results are as follows:VL17 and VL18 strains had the most significant difference in colony diameter at 34 degrees,VL18 strain was 63.16%larger than VL17.Under the condition of 37 degrees,all of the two strains were stopped growing.The inhibition of spore germination of two strains was highest at 37 degrees,the spore germination rate of VL18 strain was 2.03 times as much as that of VL17 strain.Temperature can affect the pathogenicity of Verticillium coil to aphids.Two strains had the highest pathogenicity to aphids at 28 degree,the VL18 strain was 1.02 times as much as the VL17 strain.Two strains had minimal pathogenicity to aphids at 37 degree,the VL18 strain was 2.67 times as much as the VL17 strain.The results of the study on the temperature of the aphid killing activity,mycelial growth and spore germination of Verticillium lecanii were studied.The results showed that VL17 strain was more resistant to high temperature stress than VL18.After 15,30,60,120 minutes of high temperature stress at 45 degrees,in the range of 15-60 minutes,the relative expression amount of HSP70 and SHSP gene of VL17 strain increased with the increase of stress time.The expression of SHSP gene was 7.95 times higher than that of HSP70 at the stress of 60 minutes.When the stress time was 120 minutes,the expression of two genes was lower than that of stress for 60 minutes,the expression of HSP70 was 4 times larger than that of SHSP.When the stress was more than 30 minutes,the relative expression of HSP70 and SHSP gene of VL18 was higher.The expression of SHSP was 5.85 times as much as that of HSP70 at the stress of 30 minutes.When the stress time was 120 minutes,the difference of the expression of two genes was the largest,the expression of HSP70 was 6.41 times as much as that of SHSP.The results showed that SHSP gene had a more important effect on the resistance to high temperature stress,and it was the key heat shock protein gene of Verticillium lecanii.By cloning the SHSP gene,the genomic DNA sequence was compared with the transcriptome sequence,and the SHSP gene was found to contain no introns.The knockout gene of SHSP gene was obtained by the construction of knockout vector pCAMBIA-barl-hsp and Agrobacterium mediated transformation.Screening and identification of PCR,and finally get the knockout strains.The biological characteristics of the original strains and the knockout strains were compared.The results showed that the diameter of the colony was smaller than that of the original strain,and in the same nitrogen source medium,so too.The inhibition rate of mycelial growth was higher than that of the original strain at the same time,and the spore germination rate of the knockout strain was lower than that of the original strain with the same chemical stress.The results showed that knockout strains were more sensitive to chemical stress.Under the high temperature stress of 45 degrees,with the increase of stress time,the spore germination rate of the original strains and knockout strains decreased gradually.Under the same stress condition,the spore germination rate of knockout strains was lower than that of the original strain.When the stress time was 120 minutes,the spore germination rate was 3.52 times as much as that of the knockout strain.Under UV irradiation,with the increase of irradiation time,the germination rate of the original strains and the knockout strains decreased gradually,and at the same time,the spore germination rate was higher than that of the original strain.When the stress reaches 10 minute,the spore germination rate of the original strain and the knockout strain was the biggest,and the original strain was 2.83 times as much as that of the knockout strain.Under the same number of days,the pathogenicity of the isolates was weaker than that of the original strain,By fifth days,the original strain had a significantly higher mortality rate than that of the knockout strain of aphids by the time of day 7.53%.By comparing the biological characteristics of the original strains and the knockout strains,it was found that the deletion of SHSP gene led to the decrease of the utilization of carbon and nitrogen sources,the decrease of the resistance to chemical stress,and the decrease of the ability to resist high temperature and ultraviolet radiation,and the decrease of the pathogenicity of aphids.