Cloning And Function Researching Of The Trichoderma Harzianum SOD Gene

Trichoderma. Harzianum was an antimicrobial strain. It had important applied value in the biocontrol of plant diseases, especially diseases infected through soil. T. harzianum biologically controlled the plant pathogenic fungi by mycoparasitism, bactericidal efficacy, antibiotic effect, protease effect and the competitive mechanism with space and nutrition. It was widely used in biocontrol of plant disease now. But because of various adverse suvivial conditions, the antimicrobial efficacy of T. harzianum was restricted and badly damaged, so it was necessary to enhance the adversity resistance of T. harzianum in order to realize its function of biocontrol.Express vector pCA-GSOD for filamentous fungi transformation mediated by Agrobacterium tumefaciens was successfully constructed by using binary vector of pBI121,pCAMBIA1301 and clone vector of pUC18. Including the SOD gene, hygromycin resistance gene as selected marker and CaMV35S promoter with section of T-DNA.the vector pCA-GSOD could be used for genetic transformation of mycelial fungus. The construction of vector pCA-GSO, suggests a stratergy for the enhancement of SOD expression in T. harzianum and improvement of T. harzianum'environmental resistance.In this work, genetic transformation system mediated by A. tumefaciens was used to transformate T. harzianum and may solve the problem of traditional fungus transformation, complex operation, low transformation frequency and genetic unstability of transformants faced by traditional fungus transformation system. The best conditions of this system to transformate T. harzianum were got by analysis every transformation factors. Positive transformant was harvested by the selected method, southern blot analysis and passage culturing.Antifungal activities of the transformants of T. harzianum was tested by means of dual culture with three kinds of plant pathogens, and the results showed that there was no obvious difference in inhibition activity to these pathogens between original strain of T. harzianum and its transformants obtained by A. tumefaciens. The main antifungal mechanisms of the transformants were competition and parasitism, which were the same to that of wild strain. These results indicated that our transgenic operation didn't decrease its antifungal ability. Then stress experiment of wild T. harzianum and its transformants was tested. The result indicated that transformants still suvived. Moreover, dual cultured with plant pathogens, the transformants alse suvived well; however, wild T. harzianum could not tolerate the stresses and lack the inhibition activity to these pathogens.This expression of SOD gene in T. harzianum enhanced the resistance of T. harzianum, ensured its excellent biocontriol function in various adverse conditions so as to widen the application area of T. harzianum.