Studies On Genic Function Of Pokeweed Antiviral Protein â…  And â…¡ And Antiviral Proteins Isolation From Leaves Of Pokeweed

Pokeweed antiviral protein belongs to Ribosome-inactive Proteins (RIPs). PAP has potent antiviral activity against many plant virus. pT1 was used as the template to amplify the deleted mutant PAP gene (N-deleted and N-,C-deIeted) by PCR and then the two genes were cloned into expression vector. Different IPTG-inducible expression vector containing different deleted mutant PAP gene was constructed and transferred into E.coli strain BL21 (DE3)-plysS. The two specific proteins were produced after induction and their molecular weight were 32kD and 29kD. Activity detection tests revealed that the two proteins could inhibit TMV invasion with high efficiency. The deleted mutant PAP gene was also cloned into plant expression vector pBI121 and transformed into tobacco using leaf-disc method mediated by the Agrobacterium tumefaciens. Transgenic tobacco plants were obtained through screening with kanamycin. The transgenic tobacco plants could delay TMV infection for about 25 days compared with non-transgenic tobacco plants.Pokeweed antiviral protein II (PAPII) is expressed with high level in summer leaves. The expression of PAPII is regulated by season. The total RNA was extracted from pokeweed (Phytolacca americana L.) leaves in summer using the method of TRIzol and used as template to amplify the PAPII gene by RT-PCR and then the gene was cloned into E.coli expression vector and secreted expression pPIC9K vector. The two vectors with PAPII gene were then transferred into E.coli strain BL21 (DE3)-plysS and Pachia pastor is GS115 strain respectively. The specific protein was produced induced by IPTG and methanol. SDS-PAGE results showed that only one protein band existed by induction. Activity detection tests revealed that the two proteins could inhibit TMV invasion with high efficiency.There are some isoforms of PAPs showing inhibitory ability to virus infection in various organs and at different stages of development. Three different species of the protein can be isolated from leaves of pokeweed (PAP, PAPII and PAPIII). Proteins were precipitated with 50-80% ammonium sulfate from leaves in spring, summer and autumn respectively. Proteins were purified on SP cation-exchange then on MQ anion-exchange and finally on MS cation-exchange or on MQ anion-exchange. We found that some new pokeweed antiviral proteins separated from these leaves were different from PAP, PAPII and PAPIII. But these new proteins need to be separated, purified and studied further.Five different species of the protein can be isolated from leaves, seeds and root cultures.In the present study, none was isolated from root. The nucleotide sequences of the PAP gene and PAPII gene in the root of Phytolacca americana were determined by reverse transcription polymerase chain reaction (RT-PCR) and PCR. The gene comprises 942bp and 933bp respectively, encoding 313 amino acids and 310 amino acids. When PAP gene and PAPII gene sequences in the root were compared with those in the spring leaves and summer ones, the highest similarity 100% and 99.8% were observed respectively. They both had one open reading frame (ORF). Analysis of the two sequences showed, in fact, they belonged to one species.