Construction Of Plant Expression Vector With Mutation Gene Of Pokeweed Antiviral Protein And The Study Of Its Genetic Transformation In Rice

Viral disease which influences the output and quality of rice is a serios problem in provisionment.Tranditional approaches like crop-dusting to defend this desease shall make environmental-polluting.At present, cultivating virus resistant rice by genetic engineering is one of the methond to solve this contradiction.Pokeweed antiviral protein (PAP) from the spring leaves of Phytolaccaamericana L is naturally occurring RNA-depurinating enzyme with broad-spectrum antiviral activity. There are numerous reports of mature PAP and mutations conferring some level of viral disease resistence when transformed into particular plants, and in some clinical case PAP have to be found effective to animal virus including HIV.PAP has become an important agent in agriculture and medicine.The native full-lenth PAP gene codes for 313 aminos acids.However, during posttranslational modification, 22 animo acids (singal peptide) from the N-terminus and 29 animo acids from the C-terminus are cleaved leaving a mature PAP of 262 amino acids. The C-terminal deletion mutant, PAPc, which loses 25 amino acids near C-terminus comparing to the mature PAP, has lower cytotocity and shows risistence to virus. And these 25 amino acids are necessary for toxicity but not for antiviral activity.Another mutation, PAPy, replacing Tyr-123 with Ala (Y123A) causes lower level of depurination and cytotoxicity but retains antiviral activity. This non-toxic mutation also accumulated stable levels of prote in transformants and showed risistence to virus in vitro.In the experiment, the full code sequence of PAP cDNA gene was cloned by RT-PCR from the spring leaves of the Phytolaccaamericana L. Then,the mature PAP and the two non-toxic mutations clonded by PCR using deletion method and site-directed mutagenesis.The IPTG-inducible expression vector containing the PAP genes was constructed and transformedinto E.coli BL21(DE3)plysS.The specific protein was produced,revealing that these genes has been actually achieved and exactly expressed in E.coli.The mature PAP and the non-toxic mutantions of PAP was also cloned into plant expression vector pCAMBIA-1302 and then the recombinant vector was transformed into Agrobacterium strain LBA4404.After that the genes was transformed into tobacco(Nicotiana glutinosa) using leaf-disc method by the Agrobacterium tumefaciens.The transgenic tobacco plants were measured by PCR.The non-toxic mutantion PAPy gene was transformed into rice (Oryza stiva. L) by the Agrobacterium tumefaciens in order to improving disease resistance to viral disease.Many transgenic calli and regenerations were obtained and screened by PCR. Southern bloting and northern bloting will be used to test these transformations in the future.