Isolation And Function Identification Of A Novel RNA-dependent RNA Polymerase Gene, GhRdRP, In Cotton (Gossypium Hirsutum L.)

The RNA-dependent RNA polymerase (RdRP) was discovered in Chinese cabbage 3 decades ago, since then, it has been found in several different plant species such as cauliflower, tobacco, tomato, cowpea, cucumber, rice wheat and corn. RdRP in plants can synthesize short complementary RNA with cellure and virus RNA as template, being concerned with dsRNA sequence special induced RNA silencing and playing an important role in antiviral defense. In this thesis, we select the economically important species cotton (Gossypium hirsutum) as the experiment material, and a series of studies have been conducted on the isolation, sequence and expression analysis, function identification of cotton RdRP, which can greatly help to study the function and mechanism of RdRP. The main results are as follows:1. A novel gene, termed Gossypium hirsutum RdRP (GhRdRP), was isolated from cotton. The full-length cDNA of GhRdRP is 3672 bp long, and encodes for a 1110 amino acid protein that contains all the RdRP conserved function domain.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 GhRdRP shared high identity with the RdRP in Arabidopsis, tobacco, tomato and Hordeum vulgare. The homology is from 59.29% to 66.37%. Sequences homologous to GhRdRP were also observed in yeast, Neurospora crassa, and elegans. Four introns were found in the region of genomic sequence, and more interestingly, one intron was located in the 5′UTR. Southern blot analysis showed that GhRdRP was a single copy gene in cotton genome. 2. One 954 bp promoter sequence was obtained using reverse PCR. Some regulatory elements were found in the promoter region, for example: SA, MeJA, ABA, auxin; some light responsive elements such as ACE, Box4, BoxI, G-box, I-Box; two HSE elements. Semi-quantitative RT-PCR revealed that the transcripts of GhRdRP accumulated markedly when the cotton seedlings were subjected to abiotic stimuli such as SA (salicylic acid) and 5-ssal (5-Sulfo-salicylic acid); Furthermore, GhRdRP was upregulated by Rhizoctonia solani Kuhn as pathogen attacks. These results indicate that the GhRdRP may play an important role in the plant pathogen attack response.3. Construct a sense expression vector pBI121-GhRdRP, and transformed it into the tobacco plant (NC89). 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 GhRdRP 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 separate rule of 3:1. After inoculated with Tobacco mosaic virus (TMV), Cucumber mosaic virus (CMV) and Potato virus Y (PVY), the transgenic plants exhibited higher resistence to TMV than the nontransgenic plants, but showed no difference to CMV and PVY.5. A 222 aa 3′fragment of GhRdRP was selected to construct an E.coli expression vector pET-GhRdRP-222, and then it was induced by IPTG to express in E.coli strain BL21 (DE3). The strong induced fusion protein bands were collected into PBS solution and immuned smart mouse to obtain antiserum. The value of antibody reaches 1:1000.