American Journal of Potato Research

, Volume 85, Issue 3, pp 219–226

Structure of Two Solanum bulbocastanum Polyubiquitin Genes and Expression of Their Promoters in Transgenic Potatoes

  • David R. Rockhold
  • Sophie Chang
  • Nathan Taylor
  • Paul V. Allen
  • Kent F. McCue
  • William R. Belknap
Article

Abstract

Two polyubiquitin genes, bul409 and bul427, were isolated from a Solanum bulbocastanum BAC library. The bul409 and bul427 genes encode hexameric and heptameric polyproteins, respectively. bul427 exhibits a number of features suggestive of a pseudogene: (1) The last ubiquitin monomer of bul427 is interrupted by a frame shift mutation. (2) The coding sequence is flanked 3′ by mitochondrial and chloroplast sequences and 5′ by a protein kinase pseudogene. However, characterization of cDNAs amplified using bul427-based primers demonstrated that bul427 is transcriptionally active. Chimeric transgenes encoding β-glucuronidase (GUS) translationally fused to the first ubiquitin-coding units of bul409, a truncated version 409s, and bul427 were constructed and introduced into potato. In transgenic potato lines both S. bulbocastanum promoters were weakly transcribed in tubers and efficiently transcribed in leaves. In leaves, bul409s was wound-induced, while bul427 was not. In tubers both promoters were wound-induced. In unwounded leaves and tubers, the steady state mRNA levels from both bul promoters were lower than the steady state mRNA levels from the Cauliflower Mosaic Virus 35S promoter. However, in the leaves and tubers of many of the transgenic lines the GUS activity was significantly higher in the bul lines compared to the 35S lines. The apparent inconsistency of higher enzymatic activity correlated with lower steady state levels of mRNA demonstrates the enhanced protein expression observed with ubiquitin fusion proteins.

Keywords

β-glucuronidase Intron Leaf Tuber Wound-induced 

Abbreviations

GUS

E. coli β-glucuronidase

BAC

bacterial artificial chromosome

UTR

untranslated region

Resumen

Dos genes de poliubiquitina, bul409 y bul427, fueron aislados de una librería BAC de Solanum bulbocastanum. Los genes bul409 y bul427 codifican poliproteínas hexámera y heptámera, respectivamente. El bul427 exhibe varios rasgos que sugieren un seudo gen: (1) El último monómero de bul427 es interrumpido por una mutación con desplazamiento de la pauta de lectura. (2) La secuencia codificante es flanqueada en 3′ por secuencias mitocondriales y de cloroplastos y en 5′ por un seudogen de proteína quinasa. Sin embargo, la caracterización de cDNAs amplificadas usando iniciadores basados en bul427 demostró que bul427 es transcripcionalmente activo. Se construyeron e introdujeron en papa transgenes quiméricos que codifican β-glucuronidasa (GUS) translacionalmente fusionados a las primeras unidades que codifican ubiquitina de bul409, una versión truncada de 409s y bul427. En las líneas de papa transgénica de S. bulbocastanum, los promotores fueron débilmente transcritos en los tubérculos y eficientemente transcritos en hojas. En hojas el bul409s fue inducido por heridas, mientras que bul427 no fue. En tubérculos, ambos promotores fueron inducidos por heridas. En hojas y tubérculos sin herir, los niveles mRNA en estado de equilibrio para ambos promotores bul fueron más bajos que los niveles de mRNA en estado de equilibrio del promotor 35S del virus del Mosaico de la Coliflor. Sin embargo, en hojas y tubérculos de muchas líneas transgénicas, la actividad de GUS fue significativamente más alta en las líneas bul en comparación con las 35S. La aparente inconsistencia de una actividad enzimática mayor, correlacionada con bajos niveles de mRNA en estado de equilibrio demuestra el incremento de la expresión de proteína observada con proteínas fusión ubiquitina.

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Copyright information

© Potato Association of America 2008

Authors and Affiliations

  • David R. Rockhold
    • 1
  • Sophie Chang
    • 1
  • Nathan Taylor
    • 1
  • Paul V. Allen
    • 1
  • Kent F. McCue
    • 1
  • William R. Belknap
    • 1
  1. 1.United States Department of Agriculture, Agricultural Research ServiceWestern Regional Research CenterAlbanyUSA

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