Characterization and Promoter Analysis of a Cotton RING-Type Ubiquitin Ligase (E3) Gene

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 338 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 C(3)H(2)C(3)-type RING protein. Blast searches show that GhRING1 has the highest homology to At3g19950, a zinc finger family protein 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 days post-anthesis (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 activated 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. The expression pattern of GhRING1 suggests that protein ubiquitination and turnover may be involved in transition to different stages of cotton fiber development.

Genome-wide identification and expression analysis of Gossypium RING-H2 finger E3 ligase genes revealed their roles in fiber development, and phytohormone and abiotic stress responses

“…The overexpression of rice SUMO E3 ligase gene OsSIZ1 in cotton improved fiber yield (Mishra et al 2017). Similarly, GhRING1 transcript levels were significantly elevated in elongating fibers at 15 DPA (Ho et al 2010). Our findings together with these previous findings, demonstrate the suitability of G. hirsutum RH2FE3 genes for quantitative studies at different cotton fiber developmental stages.…”

“…E3 ligases have roles in fiber cell elongation. For example, in Gossypium hirsutum (upland cotton), high transcript level of GhRING1 was observed at 15 days post-anthesis in cotton fiber and GhRING1 encodes an E3 ligase (Ho et al 2010).…”

Characterization of a Novel RING-Type Ubiquitin E3 Ligase GhRING2 Differentially Expressed in Cotton Fiber

“…The three genes were all expressed in fiber in a developmentally regulated manner. The E. coli over-expressed GhRING1 was found to have the ubiquitin E3 ligase activity by an in vitro reconstitution assay [21]. These data suggested that cotton fiber growth and development are regulated via protein ubiquitination and protein ubiquitination may play a role in fiber differentiation.…”

“…The differential expression of two E2 genes, GhUBC1 and GhUBC2 [20], and the GhRING1 [21] and GhRING2 genes in cotton fiber cells strongly suggests that cotton fiber growth and development may be regulated by protein ubiquitination. Hovav et al [38] and Al-Ghazi et al [39] have shown that several hundred genes involved in the ubiquitin-mediated protein degradation pathway, including genes for the subunits of proteasome, E2, SCF F-box proteins, and RING-type ubiquitin E3 ligases were highly up-regulated in fiber at the elongation and the secondary cell wall synthesis stages.…”

“…We previously cloned two cotton E2 genes (GhUBC1 and GhUBC2) and one E3 gene (GhRING1 encoding a RING-type ubiquitin ligase), and studied their expression in cotton fiber [20] [21]. The three genes were all expressed in fiber in a developmentally regulated manner.…”