Isolation And Functional Analysis Of A Novel Rna-dependent RNA Polymerase 6 From Nicotiana Glutinosa

The activity of a plant RNA-directed RNA polymerase (RDR) was detected 35 years ago in a search for enzymes that catalyse the replication of plant RNA viruses. It turns out that virus genomes are not amplified by plant RDRs but by virus encoded RNA-dependent RNA polymerases (RdRps). And these discoveries lead RDRs being the hot field. Since then, it has been found in several different plant species,such as Arabidopsis, tobacco, tomato, cowpea, cucumber, rice and so on. RDRs are encoded by multigene families. Six RDRs have been found in Arabidopsis and five RDRs have been isolated in rice. In this work, we select the Nicotiana glutinosa as the experiment material, and a series of research have been managed on the isolation, sequence and expression profile analysis, and functional identification of NgRDR6, which can greatly help to study the function and mechanism of RDR. The main results are as follows:1. A novel RDR gene was isolated from Nicotiana glutinosa, named as NgRDR6, (FJ490363) using RT-PCR and RACE-PCR. The full-length cDNA was 3,921 bp in size and contained an open reading frame (ORF) of 3,594 bp. There was a 123 bp 5′untranslated region (UTR) upstream of the start codon and a 204 bp 3′UTR following the stop codon. The deduced NgRDR6 protein was 1,197 aa along with a calculated molecular weight (MW) of 135.472 kDa. The result of sequence analysis of NgRDR6 at protein level showed that NgRDR6 had high homology with other RDR6 such as OsRDR6 and AtRDR6 (57.92% and 65.00% identity, respectively). And the result of phylogenetic analysis revealed that NgRDR6 had closer relation with plant RDR6 than other RDRs including RDR2s and RDR1s. It can also be found that NgRDR6 was clustered with the dicotyledonous RDRs such as OsRDR6 and NbRDR6. Moreover, the genomic DNA of NgRDR6 was 5,696 bp in length, including two introns. The two introns were 1,140 bp and 644 bp, respectively. Interestingly, intron 1 of NgRDR6 is located in the 5′UTR, which is different from other identified plant RDR6s.2. The expression profile of NgRDR6 under biotic and abiotic stresses was studied by semi-quantitative RT-PCR. And the results indicated that the NgRDR6 mRNA accumulation could be induced by ABA, GA, MeJA, CMV, Rhizoctonia Solani and Colletotrichum nicotianae. In contrast, the expression level of NgRDR6 exhibited no remarkable difference under the treatments of PVY, TMV, H2O2 and SA. Further investigation suggested several potential cis-acting elements were found in the 5′-flanking sequence of NgRDR6, which might be responsible for the enhanced response to phytohormones.3. A sense expression vector pBI121-NgRDR6 was conducted, and was transformed into the Nicotiana benthamiana. The empty vector was also transformed into tobacco at the same time as control. We carried out PCR and semi-quantitative RT-PCR identification on all obtained transgenic plants, and found that NgRDR6 has been expressed successfully in the tobacco plants.4. We analyzed the biological function of the T1 generation plants. On the basis of the molecular identification, the T1 transgenic plants heredity is consistent with the law of segregation. In the analysis of antiviral defense, the transgenic plants only exhibited higher resistence to PVY than the wild-type plants.