Home | About NASC | Address/Staff
Ask a question | Help

NASC

The European Arabidopsis Stock Centre

Belfield_MtHPL_collection

Donated by

  • Rod Casey Department of Crop Genetics, John Innes Centre
  • Eric Belfield Department of Crop Genetics, John Innes Centre

Click here to view all 15 of these lines.

Description

This collection of 15 Arabidopsis thaliana Col-0 transgenic lines include: six lines over-expressing Medicago truncatula hydroperoxide lyase E (a 13 hydroperoxide lyase) either with (+) or without (-) a putative N-terminal regulatory sequence ; six lines over-expressing Medicago truncatula hydroperoxide lyase F (a 9/13 hydroperoxide lyase) either with (+) or without (-) a putative N-terminal regulatory sequence and three lines vector only controls (L700). L700 is a light specific promoter empty cassette.

Three independent T3 generated lines for each hydroperoxide lyase construct (E/F, +/-). Homozygous for a single transgene locus on the basis of segregation patterns.

Construct and transformation details

A. thaliana ecotype Columbia (Col-0) were transformed with Agrobacterium tumefasciens (GV3101) using the floral dip infiltration method.

A modified pGPTV-BAR binary vector, pL700, was used for A. thaliana transformation. MtHPL-E and MtHPL-F cDNAs were cloned into the Gateway (Invitrogen) compatible pDESTL700 plasmid; the uidA gene was excised from the pGPTV-BAR binary vector with HindIII and SstI and replaced with potato light-inducible tissue-specific ST-LS1 gene promoter (bases 73-1574 of accession X04753). In addition, a polylinker with the BlnII, SmaI, SpeI, XhoI, and BsiWI restriction sites was added between the promoter and the nos terminator. The pL700 vector was made into a Gateway (Invitrogen) destination vector (pDESTL700) by ligating the Gateway cassette A (attR1: chloramphenicol resistance gene: ccdB negative selection gene: attR2) into the SmaI site. Restriction enzyme digest analysis indicated the cassette was clned in the correct orientation. The pL700 binary vector was used as an 'empty vector' plant transformation control.

References

  • Belfield, E.J. et al. 2007. The Gateway pDEST17 expression vector encodes a -1 ribosomal frameshifting sequence. Nucleic Acid Research 35(4): 1322-1332. PMID. 17272299.