The Transformantion And Characterization Of Mycoparasite Related Gene For Clonostachys Rosea

Clonostachys spp. species were a group of fungi inhabited widely and commonly in the soil and were important biocontrol agents against crop pathogenic fungi. The67-1strain of C.roseum was isolated from the soil collected from Hainan Province,China. Previous studies indicated that this fungus was a strong parasite and pathogen of many plant pathogens.A42kDa endochitinase gene(ThEn42) cloned from the genomic DNA of Trichoderma harzianum by plasmid PxxS1was transformed into the protoplasts of the fungus C.roseum67-1using the method of restriction enzyme-mediated integration (REM1). It was shown that the number of the7sporulation of the transformants and the amount of growth6transformants are markedly increased, enzymatic determination has found1/10of the chitinase activity is higher than wild strain,The confrontation experiment shows that1/5of the transformants, the bacteriostatic efficacy has increased.These results preliminary screen out that TC、TC-64、TC-298、TC-406、TC-514are of a more biological control potential and excellent biological control transformants,The determination control effect of soybean Sclerotinia test shows that TC-8、TC-64、TC-298has better prevent disease efficacy than67-1original strain and chemical pesticides. Expression Analysis of Chitinase gene ThEn42from C.roseum67-1transformants and wild type by Realtime-PCR,we found that the expression level of transformants TC-64is10times higher than wild type.By using protoplast conversion method to prepard the converter of Gliocladium roseum67-18-3gene,24of the transformants have been obtained through the hyg-resistant plates prescreening and PCR affination, The bioassay indicated the number of the1sporulation of the transformants and the amount of growth2transformants are markedly increased, The confrontati on experiment shows that1/4of the transformants, the bacteriostatic efficacy has increased. Further biocontrol studies with the8-3transformants and different phytopathogens will allow us to assess the value of these new strains for biological control and will provide a better understanding of the role of8-3in mycoparasitism.