美洲商陆抗病毒蛋白基因在毕赤酵母中的表达-植物病理学专业毕业论文.docxVIP

ABSTRACT1)lant ABSTRACT 1)lant virus disease is one primary crop disease．It distributes wildly and does great harm to economy of agriculture yield．People search after ways to prevention and c1．1re virus diseases for long time but not develop effective reagent yet．Many studies revealed that pokeweed antiviral protein(PAP)Can control plant virus effectively．But PAP in nature is limited which could not apply cosmically．Using bioreacter to produce PAP is one efficacious way to get a mass of PAE Someone tried this way but the yield is low．Francis expressed truncated mutant pokeweed antiviral protein gene in Pichia pastoris．The yield reached 10-12mg／L．Dr．Chen ding hu in plant virus group ofInstitute ofplant protection of the CAAS(Chinese academy of agriculture sciencesl selected PAP Muts recombinants and wanted to produce PAP through fermentation to prevention and cure plant virus disease． But as to Muts recombinants．the periods of fermentation is too long and the efficiency of using methanol is low．So using Mut5 recombinants to produce PAP in factory is not felicitousness． Linearized the expression vector pPIC9K—P including the truncated mutant pokeweed antiviral protein(PAP)gene by restriction endonuclease Sal 1 and transformed it electrically into Pichia pastoris GSll5．Mut十recombinants were selected by PCR and hiIgh yield Mut+recombinant was picked out by double film immunoblotting． To find out the difference between Mut+and Muts recombinants we compared their expression of pokeweed antiviral protein in the same conditions．SDS—PAGE results showed that there was a clear target protein band in Mtit+recombinant supernatant after 48 hours of culturing，while a faint band only in Muts recombinant after 72 hours． Western-blotting result showed that there was no remarkable difference of yield between Mut+and Muts recombinants after 6 days induced．Anti—virus activity tests revealed that culture supematants of Mut+and Mut8 recombinants could inhibit TMV infection with high effic