Establishment Of A Transformation System Of Ganoderma Linzhi And Overexpression Of Immune Protein LZ8

Ganoderma lingzhi Sheng H. Wu, Y. Cao&Y.C. Dai is Is China’s famous medicinalfungus. But, as a kind of high nutrition and health value of large basidiomycete fungi, G.lingzhi also lacks a genetically modified method and efficient system of genetically modified.This paper established by PEG mediated G. lingzhi genetic transformation system, and on thebasis of this system by adjusting immune protein expression of the system is established. Firstcloned mushroom GDP promoter successful build to pCAMBIA1302expression vector withGFP green fluorescent protein, the conversion of G. lingzhi protoplast, stability oftransformation, so as to establish efficient G. lingzhi expression system. On this basis andcloned from pleurotus ostreatus GDP promoter and G. lingzhiLZ8purpose gene clone, NOSterminator, successfully built into pSB130expression vector, G. lingzhiprotoplast, get into theson.The following results were obtained:1.The optimum conditions for the protoplast regeneration are: strain age is4d, theenzymatic hydrolysis time is3h,the temperature is31°C, pH5.0, osmotic stabilizer is0.4mol/mL2.The optimum conditions for the protoplast regeneration are：mannitol the solid MYG isused as regeneration medium.3. Establishment of binary vector pCAMBIA1302，replace the expression promoter35sin binary vector pCAMBIA1302by promoter GPD.4.Transfer the recombinant vector pCAMBIA1302into protoplast ofG. lingzhi, thenmeasuring the PCR product of transformant of G. lingzhiand observing by Fluorescencemicroscopy prove that the GFP green fluorescent protein has been transferred into G. lingzhi.5. Establishment of binary vector pSB130,theGPD promoter, LZ8target gene and NOSterminator are connected to the expression vector pSB130.6. Transfer the recombinant vector pSB130into protoplast of G. lingzhiby PEG method.G.lingzhi transformants by PCR assay and real-time PCR LZ8protein has been transferred to theG. lingzhi.