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Ocampo 210 bis, predio CCT
Rosario, Santa Fe
Soybean (Glycine max) was modified to express sunflower
(Helianthus annuus) homeobox-leucine zipper protein 4
(HaHB4) gene to confer tolerance to abiotic stresses, such
as drought and hypersaline conditions. In transgenic
Arabidopsis thaliana experiments, the plants expressing
HB4 survived in water-limited conditions, while wild-type plants
Note: The Streptomyces hygroscopicus bar gene was also
inserted into the genome and was used for selection during
transformation (not with the intention for herbicide resistance).
The term Recipient organism refers to an organism (either already modified or non-modified) that was subjected to genetic modification, whereas Parental organisms refers to those that were involved in cross breeding or cell fusion.
Soybean cultivar: Glycine max cv. Williams 82
- Agrobacterium-mediated DNA transfer
Vegetative storage protein terminator
Phosphinothricin N-acetyltransferase gene
TEV 5' Untranslated Region
Homeodomain-leucine zipper 4 promoter
Homeodomain-leucine zipper 4 gene
Nopaline Synthase Gene Terminator
bar gene cassette (Anti-sense
The transcription of the Streptomyces hygroscopicus
phosphinothricin N-acetyltransferase (bar; PAT protein)
gene is under the control of the duplicated Cauliflower Mosaic
Virus 35S promoter and the Glycine max vegetative storage
protein terminator. To enhance translation, the Tobacco Etch Virus
5' untranslated region was included at the 5' end of the
Homeodomain-leucine zipper 4 gene cassette (Sense
Transcription of the Helianthus annuus Homeodomain-leucine
zipper 4 (HaHB4) gene starts at the HaHB4
promoter and terminates at the Agrobacterium tumefaciens
nopaline synthase gene terminator. The promoter activity is
excepted to increase in response to abiotic stresses, ethylene and
insect feeding, resulting in accumulations of HaHB4
transcript and HAHB4 protein.
HaHB4 nucleotide changes:
- Deletion of nucleotides 76 to 87
- A to G substitution at position 22 (codon change from AAA to
- G to A substitution at position 351 (silent point mutation - CAG
- A to C substitution at position 401 (silent point mutation - GCA
- T to C substitution at position 432 (codon change TTT to
- C to T substitution at position 551 (codon change CCT to
HB4 amino acid changes:
- Four amino acid deletion (residue numbers 7 to 10 - results in
numbering change in residues)
- Lysine to Arginine substitution at position 22 (K22 ->
- Phenylalanine to Leucine substitution at position 159 (F159 ->
- Proline to Leucine substitution at position 175 (P175 ->
- Southern blot, next generation sequencing and PCR analyses
indicate that the plasmid backbone was absent in the genome.
- Southern blotting and sequencing confirmed the presence of a
single, T-DNA insert.
- PCR analysis indicated that the transgene insert followed
- Bioinformatic analysis indicated that no new open read frames
were created as a result of the T-DNA insertion.
- Selectable marker genes and reporter genes
- Tolerance to abiotic stress
- the protein is expressed in the soybean forage and seeds at level
comparable to or less than other crops expressing PAT
- highest levels of protein are detected in leaf and seed
- under normal conditions, the protein was difficult to detect in
leaf and seed tissue (level below limit of detection).