Molecular cloning and characterization of an inducible RNA-dependent RNA polymerase gene, GhRdRP, from cotton (Gossypium hirsutum L.)
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The RNA-dependent RNA polymerase (RdRP) cDNA, designated as Gossypium hirsutum RdRP (GhRdRP) was cloned from cotton by rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). The full-length cDNA was 3,672 bp in size and encoded an open reading frame (ORF) of 1,110 amino acids which contained the RdRP conserved functional domain and the signature motif DbDGD. Amino acid sequence alignment indicated that GhRdRP shared the highest identity (66.37%) with AtRdRP1 and had homology with other plant, fungal, yeast and nematode RdRPs. The corresponding genomic DNA containing five exons and four introns, was isolated and analyzed. Also a 5′-flanking region was cloned, and a group of putative cis-acting elements were identified. Southern blot analysis revealed a single copy of the GhRdRP gene in cotton genome. The expression analysis by semi-quantitative RT-PCR showed that GhRdRP was induced by salicylic acid (SA), 5-chloroSA (5-CSA) and fungal infection of Rhizoctonia solani Kuhn. The cloning and characterization of the GhRdRP gene will be useful for further studies of biological roles of GhRdRP in plants.
KeywordscDNA cloning Cotton RACE RNA-dependent RNA polymerase (RdRP) Semi-quantitative RT-PCR
This work was financially supported in part by the National Natural Science Foundation of China (No.30370928 and No.30571215). We also want to express our gratitude to Dr. Guo X-S (Department of Biology and Microbiology, South Dakota State University. Brookings, SD. 57007. USA.) for critical reading of the manuscript.
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