Purification Of An Antimicrobial Protein From Seeds Of Ginkgo Biloba And Cloning Of Antifungal Gene

During the growth and development, it is inevitable for plants to be attacked by different pathogenic microorganisms in the nature. Plants have evolved all kinds of effective defense systems to prevent it from invasion. A wide array of antimicrobial peptides or proteins, produced either in a constitutional or in an inducible manner, are believed to be involved in such mechanisms. Isolation and purification of the antifungal proteins from plants become more and more important. This has been proven to be a good strategy of plant disease control since the proteins can be developed for use as a novel class of antifungal agents and as the basis for making transgenic disease-resistant plants. It is also possible that such genes are safer than induced genes when used in transgenic plants and are thus potentially more acceptable given the current public opposition to genetic modified food.This assay selects the material of the seeds in Ginkgo biloba to study. Compare three methods to purify antifungal protein from the seeds of Ginkgo biloba, the result shows that the method of AKTA chromatography system is the best. So we mainly use this method to purify protein. The rough extracts of ginkgo biloba seeds is isolated by 40% and 80% ammonium sulfate precipitation, cation exchange chromatography on CM Sepharose F.F, gel filtration chromatography on Superdex 75. Purify an antifungal protein with an apparent molecular mass of 13.3kDa. The result indicates the purified antifungal protein has the activity of inhibiting Fusarium oxysporum. The antifungal protein was appraised by peptide mass fingerprint (PMF) matched with Mascot NCBInr in database (20070504). PMF was obtained by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS).Tandem MS determined the partial sequence of this antifungal protein. It indicates that the purified protein is an antifungal protein from the seeds of Ginkgo biloba. We design the primer by homologic sequence, amplify and clone gene. We expect that we can research the antifungal mechanism by cloning and expression of gene. This assay has practical significance to obtain the germplasm source of resisting fungi and new biologic medicine. At the same time, we discuss the method of extracting RNA from the seeds with amylase and albumen.