Plant-Parasitic Nematode-Protected and Herbicide Tolerant Soybean

The soy (Glycine max) has been modified for plant parasitic nematode resistance and herbicide tolerance. The soy contains Bacillus thuringiensis Cry14Ab1 for resistance to nematode plant parasites, such as soybean cyst nematode. The protein is related to other crystal proteins and thought to act on the lining of the intestine of nematodes to confer resistance. For herbicide tolerance, a Pseudomonas fluorescens 4-hydroxyphenylpyruvate dioxygenase (HPPD) gene with point mutations in the C-terminal to allow for and enhance tolerance to HPPD-inhibitor herbicides, such as isoxaflutole and mesotrione.

Soybean variety Throne

pSZ8832

Gene expression
Two gene expression cassettes were inserted in to the soy (Glycine max) genome. The gene cassettes are present in the counter-clockwise orientation.

Transcription of Bacillus thuringiensis cry14Ab1 is under control of the Arabidopsis thaliana ubiquitin 10 promoter and the Cauliflower mosaic virus (CaMV) 35S terminator. The ubiquitin 10 promoter promotes expression of cry14Ab1 at high levels throughout all plant tissues.

Transcription of Pseudomonas fluorescens 4-hydroxyphenylpyruvate dioxygenase 9 (hppdPf-4Pa) is under control of  a CaMV 35S enhanced promoter and terminator. At the 5' end of the transcript contains a synthetic optimized peptide from Zea mays and Helianthus annuus and a Tobacco etch virus (TEV) leader sequence. The transit peptide targets the protein to the chloroplast of the cells and the TEV leader sequence enhances translation of the transcript. The enhanced promoter contains duplicated enhancer sequences and promotes high levels of expression.

Note
- Sequencing analysis indicated a single T-DNA insertion without rearrangements.
- A 21 basepair sequence corresponding to ORIpVS1 vector backbone sequence was inserted into the soy genome. This sequence does not correspond to the antibiotic resistance gene.
- The T-DNA insertion resulted in a 63 basepair deletion in the soy genome.
- Bioinformatic analysis suggested the T-DNA was inserted into the 3' untranslated region of putative BON1-associated protein 1-like protein on chromosome 7.
- The coding sequence of hppdPf-4Pa contains changes that result in the following amino acid substitutions in the HPPD protein:
-- glutamic acid residue was changed for a proline residue at position 335;
-- a glycine residue for a tryptophan residue at position 336;
-- a lysine residue for an alanine residue at position 339; and
-- an alanine residue for a glutamine residue at position 340.

4-Hydroxyphenylpyruvate dioxygenase  (HPPD) is a iron (II)-dependent non-heme oxygenase that catalyzes the conversion of 4-hydroxyphenylpyruvate to homogentisate in the tyrosine catabolism pathway. Herbicides inhibiting HPPD prevent the breakdown of tyrosine resulting in stunted growth due to excess tyrosine and oxidative damage because the precursors for the photosynthetic transport chain and antioxidative systems (tocopherols and carotenoids) are not being produced.