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

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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 C3H2C3-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.

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Acknowledgments

We thank Dr. Martin Wubben for providing technical assistance in using the Nikon SMZ1000 Stereomicroscope. This study was supported by Special Research Initiative (SRI) grants from Mississippi Agricultural and Forestry Experiment Station (MAFES), approved for publication as Journal Article No. J-11733 of the Mississippi Agricultural and Forestry Experiment Station, Mississippi State University.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Mississippi State University, Mississippi State, Box 9650, MS, 39762, USA

    Meng-Hsuan Ho & Din-Pow Ma

  2. Crop Science Research Laboratory, USDA/ARS, Mississippi State, MS, 39762, USA

    Sukumar Saha & Johnie N. Jenkins

  3. Department of Plant and Soil Sciences, Mississippi State University, Mississippi State, MS, 39762, USA

    Sukumar Saha & Johnie N. Jenkins

Authors
  1. Meng-Hsuan Ho
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  2. Sukumar Saha
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  4. Din-Pow Ma
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Correspondence to Din-Pow Ma.

Additional information

The nucleotide sequence data in this article have been submitted to the GenBank Nucleotide Sequence DataBases under the accession number GU229884.

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Ho, MH., Saha, S., Jenkins, J.N. et al. Characterization and Promoter Analysis of a Cotton RING-Type Ubiquitin Ligase (E3) Gene. Mol Biotechnol 46, 140–148 (2010). https://doi.org/10.1007/s12033-010-9280-7

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