Overexpression Of Rac Gene From Trichoderma Harzianum And Preliminary Analysis Of Its Functions

Rac, a member of Rho superfamily, exists in the signal transduction pathway ofcell and plays an important role in many filamentous fungi. Trichoderma harzianum,which shows strong inhibition upon phytopathogen in many ways, is a typicalfungus that has an effective function of biocontrol. Since the signal transductiongenes play a crucial role in the pathway of biocontrol fungi when they inhibit thephytopathogen, researches of the factors in the signal transduction pathway arecontributed to reveal the biocontrol mechanism.This study aimed to analyze the function in Trichoderma harzianum T88by themethod of overexpressing rac gene. In order to amplify Rac gene, a pair of primerswhich have Nco I and BstE IIenzyme sites were used in the program of PCR. Toconstruct the overexpression plasmid, Rac gene and expression vectorpCAMBIA1301were both digested by Nco I and BstE II, and then connected by T4DNA ligase to build pCAMBIA1301:rac, To construct the overexpression plasmid,Rac gene and expression vector pCAMBIA1301were both digested by Nco I andBstE II, and then connected by T4DNA ligase to build pCAMBIA1301:rac. Thenthe overexpression plasmid was transformed into the wild type T88byAgrobactrium tumefaciens-mediated transformation of T. harzianum, and sixtytransformants were screened using PDA media containing Hyg50μg/mL. Choosefive transformants of all sixty strains to be identified by PCR and qRT-PCR. Four ofthem were identified to be the rac gene overexpression transformant.One of the transformations was choosed to measure its growth rate and sporeproduction and the growth rate of the transformant did not change significantly incomparison with wild type, but the speed of the spore production was faster thanwild type and the number of conidium is about1.7times of the wild type. In theexperiment that confront Sclerotinia sclerotiorum, the antibacterial rate of the wildtype is59.84%, while the transformant is65.06%. In the experiment that confrontTomatoes sickle bacteria, the antibacterial rate of the wild type is62.67%, while thetransforment is66.49%. The Mycelial morphology of the transforment wasobserved with optical microscope, and found that excessive expression of rac genehad no significant effect on mycelial morphology. Enzyme activity was measuredand found that the activity of cellulose enzyme, β-glycosidase, β-1,3-glucanaseenzyme activity were increased.