Title: Characterization and Promoter Analysis of a Cotton Ring-Type Ubiquitin Ligase (E3) Gene Authors
Submitted to: Molecular Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 4, 2010
Publication Date: August 1, 2010
Citation: Ho, M., Saha, S., Jenkins, J.N., Ma, D. 2010. Characterization and promoter analysis of a cotton ring-type ubiquitin ligase (E3) gene. Molecular Biotechnology. 46:140-148. Interpretive Summary: A cotton fiber cDNA and its corresponding gene have been cloned and characterized. The gene encodes a RING-type ubiquitin ligase containing 337 amino acids. A blast search showed that it has the highest homology to At3g19950 from Arabidopsis. It is expressed in fibers in a developmental manner and reaches a maximum expression in elongating fibers at 15 days after anthesis. GUS assay shows that the promoter of At3g19950 is highly activated in leaves, roots, trichomes, and also in anthers and stigma of flowers of Arabidopsis. In contrast the GUS assay directed by the cotton RING1 promoter is only located at stipules and anthers and stigma of flowers of Arabidopsis. Thus, it is a gene that may have an important role in regulation of fiber length in cotton.
Technical Abstract: A cotton fiber cDNA, GhRING1, and its corresponding gene have been cloned and characterized. The GhRING1 gene encodes a RING-type ubiquitin ligase (E3) containing 337 amino acids (aa). The GhRING1 protein contains a RING finger motif with conserved cysteine and histine residues at the C-terminus and is classified as a C3H2C3-type RING protein. Blast searches show that GhRING1 has the highest homology to At3g19950 from Arabidopsis. Real time RT-PCR analysis indicates that the GhRING1 gene is expressed in cotton fibers in a developmental manner. The transcript level of GhRING1 gene reaches a maximum in elongating fibers at 15 DPA. In vitro auto-ubiquitination assays using wheat germ extract and a reconstitution system demonstrate that GhRING1 has the ubiquitin E3 ligase activity. The histochemical GUS assay was performed to analyze tissue specificity of the GhRING1 and At3g19950 promoters in transgenic Arabidopsis plants. The GUS assay shows that the promoter of At3g19950 is highly activiated in leaves, roots, trichomes and also in anthers and stigma of flowers. In contrast, the GUS expression directed by the GhRING1 promoter is only located at stipules and anthers and stigma in flowers. The expression pattern of GhRING1 implies that the ubiquitin-proteasome pathway plays a critical role in fiber development.