Δ6D cassette:
The Micromonas pusilla Δ6 desaturase is under the control of the Linum usitatissimum (flax) conlinin2 (CNL2) promoter and terminator. A 5' untranslated leader sequence from Tobacco Mosaic Virus (TMV) is included to promote expression of Δ6 desaturase, which is required for the conversion of omega-3 long chain polyunsaturated fatty acids α-linolenic acid to stearidonic acid. The promoter and terminator are seed-specific and thus expression is expected to be constrained to seed tissues.

Δ5E cassette:
The expression of Pyramimonas cordata Δ5 elongase is under the control of Arabidopsis thaliana fatty acid elongase (FAE1) promoter and the Glycine max lectin (LC1) terminator. A 5' untranslated leader sequence from TMV is included to promote expression of Δ5 elongase, eicosapentaenoic acid (20:5) to docosapentaenoic acid. The expression of the gene occurs during lipid accumulation during embryo development due to the specificity of the promoter.

Δ5D cassette :
The Pavlova salina delta-5 desaturase coding sequence is under control of the Brassica napus napin promoter and the Agrobacterium tumefaciens nopaline synthase (nos)  terminator. A 5' untranslated leader sequence from TMV is included to promote expression of Δ5 desaturase, which converts eicosatetraenoic acid to eicosapentaenoic acid. The Nicotania tabacum matrix attachment region from the Rb7 gene further enhances and maximizes expression and stability of all substrates in the pathway.

ω3D cassette:
Pichia pastoris delta 15/Omega-3 desaturase is under the control of the flax conlinin1 (CNL1) promoter and terminator, which result in seed-specific expression of the gene. A 5' untranslated leader sequence from TMV is included to promote expression of Δ15/ω3 desaturase, which adds a double bond to linoleic acid to produce α-linolenic acid.

Δ4D cassette:
Pavlova salina Δ4 desaturase under the seed-specific flax CNL2 promoter and terminator. A 5' untranslated leader sequence from TMV is included to promote expression of Δ4 desaturase, which between the third and forth carbon of docosapentaenoic acid (22:5) to produce docosahexaenoic acid (22:6).

Δ12D cassette:
Expression of the Lachancea kluyveri Δ12 desaturase gene is under control of the flax CNL1 promoter and terminator for seed-specific expression. Expression is enhanced by a 5' untranslated leader sequence from TMV. [/i]Δ12 desaturase[/i] inserts a double bond of oleic acid to produce linoeic acid. The N. tabacum matrix attachment region from Rb7 further enhances and maximizes expression and stability of all substrates in the pathway.


Δ6E cassette:
Pyramimonas cordata Δ6 elongase is controlled by the A. thaliana FAE1 promoter and the G. max LC1 terminator. Expression is enhanced by a TMV 5' untranslated leader sequence.Δ6 elongase lengthens the fatty acid chain of stearidonic acid (18:4) to form eicosatetraenoic acid (20:4).

pat cassette:
The phosphinothricin N-acetyltransferase (pat) gene from Steptomyces viridochromogenes are controlled by the Cauliflower Mosaic Virus 35S promoter and the nos terminator. Due to the native high G:C content, the pat gene has been codon optimized for expression in plants.

Note:
I - The different orientations (clockwise/anticlockwise) of the genetic elements assist in improving transcription and increasing expression by including some spacing in protein transcription.

II - DHA canola was characterized with vector-targeted sequencing, whole-genome sequencing and PCR amplicon sequencing the data from which shows that DHA canola contains two T-DNA inserts in its genome and both T-DNA inserts are required for achieving the current level of DHA in the seed.

III - Whole genome sequencing confirmed that no vector backbone sequence from pJP3416_GA7-ModB, nor any genomic DNA from A. tumefaciens were present in the GM canola.

IV - The Multiple reaction monitoring quantification confirmed that none of the introduced LC- ω3-PUFA pathway enzymes were detected in 250 μg of total protein extracts from the control AV Jade in all growth stages from 5-true-leaf to mature seed, for samples collected from both field trial sites. However, all the seven enzymes were detected in DHA canola developing and/or mature seeds but not in other tested tissues including root, flower and whole plant. This is as expected because the expression of all these genes is controlled by seed-specific promoters.