American Journal of Potato Research

, Volume 88, Issue 6, pp 485–492 | Cite as

Structure of Two Solanum tuberosum Steroidal Glycoalkaloid Glycosyltransferase Genes and Expression of their Promoters in Transgenic Potatoes

  • Kent F. McCue
  • David R. Rockhold
  • Alyssa Chhan
  • William R. Belknap
Article

Abstract

The Sgt2 gene in potato encodes a solanidine glucosyltransferase and is present as two distinct alleles expressed in cultivated potatoes. Promoter regions of both steroidal glycoalkaloid biosynthetic gene alleles, Sgt2.1 and Sgt2.2, were isolated from Solanum tuberosum cv. Russet Burbank genomic DNA. The genomic sequences of Sgt2.1 and Sgt2.2 were isolated by PCR amplification using a conserved region of Sgt2 and artificial upstream primers. The longest sequences for each allele were used to create β-glucuronidase (GUS) reporter gene fusions. Fusion constructs were mobilized into stable transgenic lines for analysis of promoter expression in leaves and tubers under control and wounded conditions. S. tuberosum promoters from Sgt2.1 and from Sgt2.1 produced GUS activity in transgenic potato leaves and tubers comparable to GUS activity produced by the CaMV35S promoter. The CaMV35S promoter is a strong promoter frequently used in plant biotechnology. Both Sgt2 promoters exhibited activities similar to the CaMV35S promoter in tubers and lower relative activities in leaves. On average the Sgt2.2 promoter exhibited higher activity in both leaves and tubers relative to the Sgt2.1. There was no consistent effect of wounding on GUS activity from the Sgt2.2 promoter in leaves or tubers. The Sgt2.1 promoter supported higher transgene activity in tubers versus leaves and exhibited small but consistent increases in response to wounding in tubers only. This may be due to the presence of a MITE sequence in the Sgt2.1 promoter.

Keywords

Transgenic Sgt2 Glucosyltransferase Leaf Tuber GUS expression 

Resumen

El gen Sgt2 codifica para una solanidina-glucosiltransferasa y está presente como dos alelos distintos expresados en papas cultivadas. Las regiones promotoras de estos dos genes alelos biosinteticos de glicoalcaloides steroidales, Sgt2.1 y Sgt2.2, se aislaron de ADN genómico de Solanum tuberosum cv. Russet Burbank. Se aislaron las secuencias genómicas de Sgt2.1 y Sgt2.2 mediante amplificación por PCR usando una región conservada de Sgt2 y con iniciadores artificiales de partes superiores. Se usaron las secuencias más largas para cada alelo para crear fusiones de genes reporteros de β-glucuronidasa (GUS). Las fusiones generadas se movilizaron hacia líneas transgénicas estables para el análisis de la expresión del promotor en hojas y tubérculos bajo condiciones de control y de heridas. Los promotores de S. tuberosum de Sgt2.1 y de Sgt2.2 produjeron actividad GUS en hojas de papa transgénica y en tubérculos comparable con la actividad GUS producida por el promotor CaMV35S. Éste es un fuerte promotor que se usa frecuentemente en biotecnología de plantas. Los dos promotores de Sgt2 exhibieron actividades similares a las del promotor CaMV35S en tubérculos y actividades relativas más bajas en hojas. En promedio, el promotor Sgt2.2 exhibió mayor actividad tanto en hojas como en tubérculos en relación con el Sgt2.1. No hubo efecto consistente de heridas en la actividad de GUS del promotor Sgt2.2 en hojas o tubérculos. El promotor Sgt2.1 resistió más alta actividad transgénica en tubérculos contra hojas, y mostró aumentos pequeños pero consistentes en respuesta a heridas solamente en tubérculos. Esto pudo deberse a la presencia de una secuencia MITE en el promotor Sgt2.1.

Supplementary material

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Supplemental Fig. 1(DOCX 19 kb)
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Supplemental Fig. 2(DOCX 46 kb)
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Supplemental Fig. 3(DOCX 17 kb)

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Copyright information

© Potato Association of America 2011

Authors and Affiliations

  • Kent F. McCue
    • 1
  • David R. Rockhold
    • 1
  • Alyssa Chhan
    • 1
  • William R. Belknap
    • 1
  1. 1.USDA, Agricultural Research Service, Western Regional Research Center, Crop Improvement and Utilization Research UnitAlbanyUSA

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