Cloning And Expression Analysis Of An RNA-dependent RNA Polymerase Gene, NgRDR1, From Nicotiana Glutinosa

The RNA-dependent RNA polymerase (RDR) was discovered in Chinese cabbage three decades ago, since then, it has been found in several different plant species such as cauliflower, tobacco, tomato, cowpea, cucumber, rice, wheat and corn. The RNA-dependent RNA polymerases play a key role in RNA silencing, heterochromatin formation and natural gene regulation. In this thesis, we select the Nicotiana glutinosa as the experiment material, and a series of studies have been conducted on the isolation, sequence and expression analysis, function identification of NgRDR1, which can greatly help to study the function and mechanism of RDR. The main results are as follows:1. A novel gene, termed Nicotiana glutinosa RDR (NgRDR1), was isolated from Nicotiana glutinosa. The full-length cDNA of NgRDR1 is 3635 bp, and encodes for an 1117 amino acid protein that contains all the plant RDRs conserved function domains. One signature motif DbDGD (b is a bulky residue) which was predicted to form part of the catalytic site and contributed to catalysis via a coordinated divalent cation in this domain. Amino acid sequence alignment revealed that NgRDR1 shared high identity with the RDRs in Arabidopsis, tobacco, tomato and Hordeum vulgare. The homology is from 55% to 94%. Four introns were found in the region of genomic sequence, and more interestingly, one intron was located in the 5′UTR.2. One 1333 bp promoter sequence was obtained using LA PCR and reverse PCR. Some regulatory elements were found in the promoter region, such as HSE, MeJA (methyl jasmonate) and auxin; some light responsive elements such as AE, ATCT-motif, G-box and I-Box. Semi-quantitative RT-PCR revealed that the transcripts of NgRDR1 accumulated markedly when the tobacco seedlings were subjected to abiotic stimuli such as SA (salicylic acid), INA (isonicotinic acid), H2O2 (hydrogen peroxide) and MeJA. Furthermore, NgRDR1 expression can be up-regulated by Potato virus Y (PVY), Tobacco mosaic virus (TMV) and Cucumber mosaic virus (CMV), but not by Potato virus X (PVX). Besides, different kinds of fungi can also induce NgRDR1 expression. These results indicate that the NgRDR1 may play an important role in the plant pathogen attack response.3. Constructed a sense expression vector pROKII-NgRDR1, and transformed it into the Nicotiana benthamiana. We also transformed the empty vector into tobacco at the same time as control. We carried out PCR identification and semi-quantitative RT-PCR analysis on some transgenic plants, and found that NgRDR1 had 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 separate rule of 3:1. After inoculated with TMV, CMV, PVY and PVX, the transgenic plants exhibited higher resistence to TMV than the nontransgenic plants, but showed no difference to CMV, PVY and PVX.