Drought and Glyphosate tolerant soybean

Soybean (Glycine max) was produced through crossing Verdeca HB4 Soybean and Roundup Ready™ soybean. The stacked modifications confer tolerance to drought conditions and glyphosate herbicides through the expression of sunflower (Helianthus annuus) homeobox-leucine zipper protein 4 (HaHB4) and Agrobacterium tumefaciens 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) proteins, respectively.

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).

IND-ØØ41Ø-5 - Glycine max cv. Williams 82

MON-Ø4Ø32-6 - Line: A5403

pIND2-HB4 & PV-GMGT04

Genetic elements introduced from pIND2-HB4
bar gene cassette (Anti-sense orientation)
The transcription of the Streptomyces hygroscopicus phosphinothricin N-acetyltransferase (bar; PAT protein) gene is under the control of the duplicated Cauliflower Mosaic Virus (CaMV) 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 bar transcript.

Homeodomain-leucine zipper 4 gene cassette (Sense orientation)
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 AGA)
- G to A substitution at position 351 (silent point mutation - CAG to CAC)
- A to C substitution at position 401 (silent point mutation - GCA to GCC)
- T to C substitution at position 432 (codon change TTT to CTT)
- C to T substitution at position 551 (codon change CCT to CTT)

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 -> R18)
- Phenylalanine to Leucine substitution at position 159 (F159 -> L155)
- Proline to Leucine substitution at position 175 (P175 -> L171)

Note:
- 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 Mendelian inheritance.
- Bioinformatic analysis indicated that no new open read frames were created as a result of the T-DNA insertion.


Genetic elements introduced from PV-GMGT04
Agrobcterium tumefaciens 5-enolpyruvylshikimate-3-phosphate synthase (cp4-epsps) transcription is driven by the CaMV 35S Enhanced promoter and transcription terminates at the A. tumefaciens nopaline synthase terminator. The protein is localized to the chloroplast due to the inclusion of a Petunia hybrida chloroplast transit peptide 4 at the 5' end of the transcript. High levels of CP4-EPSPS transcript are expected due to the constitutive activity of CaMV 35S enhanced promoter.

Note:
- Southern blot and PCR analysis indicated that only a single transformation cassette containing the EPSPS coding sequence was integrated into the host genome.
- A deletion in the enhancer region of the CaMV 35S enhanced promoter occurred. However, the remainder of the E35S promoter was functional.
- Analysis also indicated that there was no integration of segments of the vector backbone, the GUS coding sequence or the CmoVb promoter.
- The expression of the EPSPS protein was confirmed by Western blotting, ELISA and EPSPS enzyme activity assay.

For more information, kindly refer to the parental LMO records.

PAT protein (from HB4 soybean)
- 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 tissue

HB4 protein (from HB4 soybean)
- under normal conditions, the protein was difficult to detect in leaf and seed tissue (level below limit of detection)

EPSPS protein
- cp4-epsps coding sequence encodes a 455 amino acid protein and results in the synthesis of the full length and functional ~46kDa protein