Cloning Of CDNA Encoding Pokeweed Antiviral Protein From Phytolacca Americana And Its Transformation Into Tomato

Pokeweed antiviral protein(PAP), isolated from leaves and seeds of Phytolacca americana, are single-chain ribosome-inactive proteins(RIPs) with molecular mass of 29 to 31KD, which can inhibit translation by catalytically removing a specific adenine residue from a highly conserved, surface-exposed, stem-loop structure in the large rRNA of eukaryotic and prokaryotic ribosomes. PAP displays broad-spectrum antiviral activity against a number of different viruses, including plant and animal viruses. The property of PAP provides a way to produce multiple-virus-resistant transgenic plants by introducing and expressing a single gene. The C-terminal deletion mutant, PAP-c, which loses 25 amino acids near C-terminus comparing to the mature PAP of 262 amino acids, has lower cytotoxicity and shows resistance to viruses. These 25 amino acids near C-terminus is necessary for toxicity but not for antiviral activity, suggesting that antiviral activity of PAP can be dissociated from its toxicity. Expression of PAP-c in transgenic plants will be safer than that of wild-type of PAP.According to the published sequence of PAP, the cDNA encoding PAP and PAP-c were obtained by using PCR, respectively. The leaf specific expression promoter rbcS-3A, which confers light-inducible and organ-specific expression, was also amplified from tomato DMA by PCR. In order to transfer PAP or PAP-c gene into tomato, three plant-expression-vectors containing PAP or PAP-c were constructed with CaMV 358 promoter and rbcS-3A promoter respectively. Agrobacterium tumefaciens containing plant transformation vectors was used to transform tomato self-line TP002 by the leaf disc method.The results of this study are as follows:1 The cDNA encoding PAP and C-terminal deletion mutant PAP-c were obtained by using PCR. The PAP cDNA has an open reading frame of 940bp, which is 99.7% homologous to the published sequence. The PAP-c cDNA has an open reading frame of 775bp, which is 99.6% homologous to the corresponding sequence of the published PAP cDNA.2 The rbcS-3A promoter sequence was amplified from tomato genome DNA by using PCR. Sequencing results showed, the 1150bp fragment obtained in this study has 99% homology to the rbcS-3A promoter sequence published in GeneBank and contains intact TATA box andCAAT box. Its enhancer region, silencer region and promoter function region are 100% homologous to those of published rbcS-3A.3 Based on pBI121, three plant-expression-vectors containing PAP or PAP-c were constructed with CaMV 35S promoter and rbcS-3A promoter respectively. The pBPAP has PAP gene expressed from CaMV 35S promoter and pBPAP-c has PAP-c gene with the same promoter. The pB-rbc-PAP-c has PAP-c gene modulated by rbcS-3A promoter from tomato.4 By Agrobacterium tumefaciens mediated transformation, PAP and PAP-c gene were induced into tomato self-line(TP002) through two different plant expression vectors, pBPAP and pB-rbc-PAP-c, respectively. Three regenerated tomato plants were proved to be transgenic plants by PCR and Southern blotting. The results of RT-PCR for transgenic plants showed, PAP gene was expressed in both leaves and fruits of No.9 transgenic plant under CaMV 35S promoter, PAP-c gene was only expressed in the leaves not in the fruits of No.6 transgenic plant under rbcS-3A promoter. To test the resistance to virus of transgenic tomato plants, they were mechanically inoculated with TMV and CMV respectively. The results indicated , comparing to the non-transformed control plants, No.5 and No.9 plants displayed resistance to TMV, No.6 plant displayed resistance to TMV and CMV.