Cloning And Expression Of Antifungal Protein And Chitinase Gene

With the popularization of greenhouse vegetables,the harm which pathogeny fungi cause is becoming serious.The employment of the traditonal chemistry pesticide inevitably bring the remaining danger.It is known to all that many microbe could be effective to restrain the pathogeny fungi since the 1960s. Among the biocontrol microbe trichoderma is very common and has economical significance.Trichoderma has serious antagonism and parasite action when comfronting the pathogeny fungi from plant.There is widespread use in the feed and food and the biological pesticide industry.The study of trichoderma has theory significance and practical application value.This work focused on genetically improving the trichoderma W512 which was separated form soil to gain better antifungal performance.We adopted the soil sample from the greenhouse around Jinan, designed specifical trichoderma screening media, separated a trichoderma having preferably biocontrol ability,identified approximately the fungi as Trichoderma by the morphologic observa tion, made phylogenetic relationship analysis on 18S rDNA sequence to identified it as Trichoderma viride, cloned the chitinase gene (ech) from W512.In this work microplate reader and 96-well flatted-bottom plate were used to measure the curve of the filamentous fungi growth, also were used to determine the antifungal efficency of the sample. We also cloned the antifunal protein gene (paf) and cDNA gene. In the Escherichia coli BL21 we expressed the PAF protein(GST-PAF,GST-proPAF,GST-mature-PAF,mature-PAF) by virtue of expression vector pBV220 and fusion expression vector pGEX4T-1. But no antifungal activity was determined.We constructed the induced fungal expression vector (pCBH) by virtue of the terminator and promoter of the cbh1 from Trichoderma reesei. We cotransformed the W512 using the plasmid and p3SR2. At present we isolated six transformants...