Characterization And Functional Analysis Of Ghrdr6, A Novel RDR6 Gene From Cotton (Gossypium Hirsutum L)

RNA-dependent RNA polymerases (RDR) have been reported for over 40 years,and was fristly discovered in chinese cabbage. Since then, it has been found in several different plant species. It has been reported that RDR in plants can synthesize short complementary RNA with cellular and virus RNA as template, which play an important role in dsRNA sequence special induced RNA silencing and antiviral defence. Six RDR (RDR1, RDR2, RDR3a, RDR3b, RDR3c, RDR6) are reported to exist in Arabidopsis thaliana. Here, we select Gossypium hirsutum L. as the experiment material, and a series of research have been conducted on the isolation, sequence and expression pattern analysis, the resistance defence of overexpressed plants and developmental analysis of GhRDR6, which can contribute to understand the function and mechanism of RDR6. The main results are as follows:(1) A novel RDR gene was cloned from cotton, named as GhRDR6, (GenBank accession number: GQ254649) using RT-PCR and RACE-PCR. The full length cDNA of GhRDR6 was 4183 bp, containing a 331 bp 5′untranslated region (UTR), a 261 bp 3′UTR, and a 3591 bp complete open reading frame (ORF) that encodes a polypeptide of 1196 amino acids with a calculated molecular mass of 138.225 kDa. The amino acid sequence of GhRDR6 was 66%, 66%, 56% identical to AtRDR6, NtRDR6, and OsRDR6 respectively, while it only shared 31% identical to GhRDR1. The GhRDR6 contains all the highly conserved sequence motifs that are presented in all RDR families. One signature motif DbDGD (b is a bulky residue) which was predicted to form part of the catalytic site, contributed to catalysis via a coordinated divalent cation in this domain. Sequence alignment between genomic DNA (GenBank accession number: GQ254651) and cDNA indicated that the GhRDR6 gene genomic sequence contains two introns, and one of them in the 5′UTR. Southern blot showed that GhRDR6 was present as a single copy in the cotton genome.(2) The expression profile of GhRDR6 in different organs and under various abiotic stresses was studied by semi-quantitative RT-PCR. The results showed that GhRDR6 displayed organs specific expression patterns and may play a role in root development. Semi-quantitative RT-PCR showed that GhRDR6 was up-regulated by application of various phytohormones including methyl jasmonate (MeJA), abscisic acid (ABA), jasmonate (JA),α-naphthlcetic acid (NAA), gibberellins (GA) and ethylene (ET). In addition, the expression of GhRDR6 was increased by the treatments of wound, cold (4°C), and NaCl, but not by drought. These data suggest that GhRDR6 may play an important role in plant defense responses.(3) A sense expression vector pBI121-GhRDR6 was conducted, and was transformed into the Nicotiana benthamiana using the A. tumifaciens-mediated transformation. The empty vector was also transformed into tobacco at the same time as control. We carried out PCR identification and Northern blot analysis on some transgenic plants, and found that GhRDR6 has been expressed successfully in N. benthamiana. 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. Moreover, we analyzed the Northern blot of the T2 generation plants. The result showed that the GhRDR6 was expressed in N. benthamiana.(4) To evaluate the function of GhRDR6 in root growth, the transgenic plants and the WT plants were grown on MS medium. The transgenic seedlings had longer roots by 1-2 cm than those of the WT seedlings when growing on MS medium for 3 weeks, but this was not significant after 6 weeks. And when seedlings of transgenic plants were transferred to MS with ABA, the related root growth increased 2-2.5 cm than WT seedlings. Similarly, the relative root growth in WT seedlings increased only 0.5-1 cm, whereas those of R1 and R6 was 1-1.5 cm and 1.5-2 cm under treatments of JA, MeJA, and GA, respectively. No obvious change could be found under 6-BA, NAA, NaCl, Mannitol, and PEG treatments. No obvious change could be found in germination of both transgenic plants and WT plants.(5) After inoculated with PVY, the transgenic plants exhibited higher resistence to PVY than the nontransgenic plants. Similar results have been obtained by the H2O2 accumulation detection through DAB staining. The CP gene expression was detected with the lowest level in R6 and the highest in WT. Furthermore, the expression analysis of CP (PVY) gene and NPR1 gene shown that virus growths in WT were greater compared with R6. NbPR1a, as the SA-dependent related gene, showed no significant differences in WT and transgenic plants, whereas the NbNPR1 gene was slightly induced in transgenic plants compared to the increased NbOsmotin, transcript accumulation was increased in transgenic plants, while the NbPR4 gene expression was decreased in transgenic than WT plants. NbACOI gene showed no difference in WT and transgenic plants.(6) The mRNA accumulation of GhRDR6 was detected after exposed to various abiotic stresses. This might result in a low-temperature tolerance of GhRDR6 overexpressing transgenic plant. After a 7-day recovery, the survival rates of transgenic seedlings were observed to be significantly higher than those of the WT. The new leaf numbers were shown more than WT seedlings. But the overexpressing transgenic plant showed no difference to the WT after drought and solt treatments.