GMO molecular characterization details

GMO detailed information

GMO:
LBFLFK
Alias:
UID:
BPS-BFLFK-2
Tradename:
Companies/Institutions:
BASF
Species:
Brassica napus
(canola, oilseed rape, rapeseed)
Traits:
EU authorisation:

General Information

Description:

LBFLFK canola was produced via Agrobacterium rhizogenes-mediated transformation using enzyme-encoding genetic sequences and associated expression cassettes that were introduced from a number of eukaryotic organisms (Phytophthora sojae, Ostreococcus tauri, Thalassiosira pseudonana, Physcomitrella patens, Thraustochytrium sp., Phytophthora infestans, Pythium irregulare, Pavlova lutheri and Arabidopsis thaliana) to alter the production of specific fatty acids in canola, resulting in the production of omega-3 LC-PUFA fatty acids and providing tolerance to treatment with the herbicide active ingredient imazamox using a single transformation vector. The expression of these proteins in LBFLFK canola allows for the synthesis of long-chain omega-3 polyunsaturated fatty acids (LC-PUFAs), including EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid), from oleic acid and for tolerance to treatment with an imidazolinone herbicide.

LBFLFK canola provides a plant-based and scalable production system for omega-3 fatty acids and will be another source of EPA and DHA for consumers as either a food ingredient or as an aquaculture feed ingredient.

Next generation sequencing of total genomic DNA and subsequent bioinformatic analysis demonstrated that LBFLFK has two inserts integrated at two separated loci (Insert1 and Insert2) and confirmed the absence of the transformation vector backbone sequences in event LBFLFK.

Sanger sequencing analyses confirmed that each of the two inserts has the intended 13 gene expression cassettes and was identical to the vector T-DNA except for two single nucleotide changes in Insert1 and one nucleotide change in Insert2 that do not impact the functionality of the proteins produced. Both T-DNA inserts were intact, apart from a short rearrangement of 64 bp in the RB sequence of Insert1. A comparison to the sequence of the insertion site from the parental canola Kumily variety demonstrated that an 8-bp deletion was created at the genome integration site of Insert1 (Locus1) and a 31-bp deletion was created at the genome integration site of Insert2 (Locus2) in Kumily. No canola genomic sequence rearrangements were found at either integration site.

Links regarding to this GMO:

USDA APHIS http://www.aphis.usda.gov/brs/aphisdocs/17_32101p.pdf,

Patent and detection method http://encrypted.google.com/patents/EP3218499A1?cl=tr,

BCH https://bch.cbd.int/en/database/record?documentID=115656

Transformation / Modification technique:

Agrobacterium-induced

Inserts / Modification

Name:	insert1_LBFLFK
Comment:	Compared to the T-DNA of the transformation vector the insert has a 184 bp truncation of the 5΄ end of the right border (RB) and a 72 bp truncation of the 3΄ end of the left border (LB). In addition the first 64 bp in the RB was determined to be a rearrangement of short T-DNA RB-derived repeats. The insertion of insert1_LBFLFK introduced an 8 bp deletion in the host genome.
Map:

Genetic Elements:

Reference: USDA-APHIS

Elements:

Order	Name	Donor	Size (bp)	Position	Definition	Trait	Comment
1	V-RB-RHIRD	Agrobacterium tumefaciens		-			truncated
2	V-intervening-SYNTH	Synthetic	180	329-508
3	P-usp-VICFA	Vicia faba	684	509-1192	Promoter of USP. Bäumlein, H. et al. (1991). Molecular and General Genetics, 225, 459-467
4	I-At1g01170	Arabidopsis thaliana	252	1193-1444	Intron-containing 5' UTR of locus At1g01170; Nakabayashi, K. et al. (2005). Plant Journal 41, 697-709
5	V-intervening-SYNTH	Synthetic	2	1445-1446
6	CS-detla6E-PHYPA	Physcomitrella patens	1173	1147-2319	delta6 elongase gene; Zank, T.K. et al (2002). Plant Journal 31, 255-268	Altered fatty acids and oils
7	T-35S-CaMV	Cauliflower mosaic virus	216	2320-2535	3' untranslated region (3' UTR) of the 35S gene from Cauliflower mosaic virus. Gardner et al. (1981). Nucleic Acids Res, 9:2871-2888
8	V-intervening-SYNTH	Synthetic	92	2536-2627
9	P-cnl1-LINUS	Linum usitatissimum	1064	2628-3691	Promoter of the conlinin1 gene. Truska et al. (2003). Plant Phys and Biochem, 41,141-147
10	I-At5g63190	Arabidopsis thaliana	377	3692-4068	Intron-containing 5' UTR of locus At5g63190; Wang, Y. et al. (2008). Plant Physiology 148, 1201-1211
11	V-intervening-SYNTH	Synthetic	3	4069-4071
12	CS-delta5D-THRSP	Thraustochytrium sp.	1320	4072-5391	delta5 desaturase gene; Qiu, X. et al. (2001). Journal of Biological Chemistry 276, 31561-31566	Altered fatty acids and oils
13	T-ocs-RHIRD	Agrobacterium tumefaciens	192	5392-5583	3' untranslated region (3' UTR) of the octopine synthase gene from Agrobacterium tumefaciens Ti-Plasmid. DeGreve et al. (1982) J Mol Appl Genet. 1(6):499-511
14	V-intervening-SYNTH	Synthetic	135	5584-5718
15	P-sbp-VICFA	Vicia faba	1799	5719-7517	Promoter of sucrose binding related protein. Grimes, H.D. et al. (1992). Plant Cell 4, 1561-1574
16	I-At1g65090	Arabidopsis thaliana	455	7518-7972	Intron-containing 5' UTR of locus At1g65090; Braybrook, S.A. et al. (2006). PNAS 103, 3468-3473
17	V-intervening-SYNTH	Synthetic	9	7973-7981
18	CS-delta6D-OSTTA	Ostreococcus tauri	1371	7982-9352	delta6 desaturase gene; Domergue, F. (2005). Biochemical Journal 389, 483-490	Altered fatty acids and oils
19	V-intervening-SYNTH	Synthetic	27	9353-9379
20	T-CDI-SOLTU	Solanum tuberosum	235	9380-9614	Terminator of cathepsin D inhibitor gene; Hannapel, D.J. et al. (1993). Plant Physiology 101, 703
21	V-intervening-SYNTH	Synthetic	78	9615-9692
22	P-pxr-LINUS	Linum usitatissimum	1727	9693-11419	Promoter of peroxiredoxin. Duwenig, E. and Loyall, L.P. (2006). Patent WO2006089950 A2, WIPO
23	I-At1g62290	Arabidopsis thaliana	846	11420-12265	Intron-containing 5' UTR of locus At1g62290 (aspartyl protease family protein); Chen, X. et al. (2002). Eur J Biochem 269, 4675-4684
24	V-intervening-SYNTH	Synthetic	13	12266-12278
25	CS-delta6E-THAPS	Thalassiosira pseudonana	819	12279-13097	delta6 elongase gene; Ambrust, E.V. et al. (2004). Science 306, 79-86	Altered fatty acids and oils
26	V-intervening-SYNTH	Synthetic	55	13098-13152
27	T-pxr-ARATH	Arabidopsis thaliana	400	13153-13552	Terminator of peroxidin. Haslekås, C. et al. (1998). Plant Molecular Biology 36, 833-845
28	V-intervening-SYNTH	Synthetic	169	13553-13721
29	P-napA-BRANA	Brassica napus	664	13722-14385	Promoter of seed storage protein napin A/B. Ellerström, M. (1996). Plant Molecular Biology 32, 1019-1027
30	I-At5g63190	Arabidopsis thaliana	377	14386-14762	Intron-containing 5' UTR of locus At5g63190; Wang, Y. et al. (2008). Plant Physiology 148, 1201-1211
31	V-intervening-SYNTH	Synthetic	6	14763-14768
32	CS-delta12D-PHYSP	Phytophthora sojae	1197	14769-15965	delta12 desaturase gene; Cirpus, P. and Bauer, J. (2006). Patent WO2006100241 A2, WIPO	Altered fatty acids and oils	sequence contains a cytosine to adenine nucleotide change which results in a phenylalanine to leucine amino acid substitution (F83L) in the delta-12D protein.
33	V-intervening-SYNTH	Synthetic	18	15966-15983
34	T-rbcS_E9-PEA	Pisum sativum	558	15984-16541	3' untranslated region (3' UTR) f the rinulose-1,5-bisphosphate carboxylase, small subunit (rbcS E9) gene. Coruzzi et al. (1984) EMBO J. 3:1671-1679
35	V-intervening-SYNTH	Synthetic	92	16542-16633
36	P-setl-BRANA	Brassica napus	1234	16634-17867	Promoter of SETL. Bauer, J. and Senger, T. (2010). Patent WO2010000708, WIPO
37	V-intervening-SYNTH	Synthetic	2	17868-17869
38	CS-omega3D-PYTIR	Pythium irregulare	1092	17870-18961	omega3 desaturase gene; Cheng, B. et al. (2010). Transgenic Research 19, 221-229	Altered fatty acids and oils
39	V-intervening-SYNTH	Synthetic	21	18962-18982
40	t-setl-BRANA	Brassica napus	614	18983-19596	Terminator of SETL. Bauer, J. and Senger, T. (2010). Patent WO2010000708, WIPO
41	V-intervening-SYNTH	Synthetic	78	19597-19674
42	P-usp-VICFA	Vicia faba	684	19675-20358	Promoter of USP. Bäumlein, H. et al. (1991). Molecular and General Genetics, 225, 459-467
43	I-At1g01170	Arabidopsis thaliana	252	20359-20610	Intron-containing 5' UTR of locus At1g01170; Nakabayashi, K. et al. (2005). Plant Journal 41, 697-709
44	V-intervening-SYNTH	Synthetic	10	20611-20620
45	CS-omega3D-PHYIT	Phytophthora infestans	1086	20621-21706	omega3 desaturase gene; Wu, G. et al. (2005). Nature Biotechnology 23, 1013-1017	Altered fatty acids and oils
46	V-intervening-SYNTH	Synthetic	8	21707-21714
47	T-35S-CaMV	Cauliflower mosaic virus	216	21715-21930	3' untranslated region (3' UTR) of the 35S gene from Cauliflower mosaic virus. Gardner et al. (1981). Nucleic Acids Res, 9:2871-2888
48	V-intervening-SYNTH	Synthetic	135	21931-22065
49	P-setl-BRANA	Brassica napus	1234	22066-23299	Promoter of SETL. Bauer, J. and Senger, T. (2010). Patent WO2010000708, WIPO
50	V-intervening-SYNTH	Synthetic	2	23300-23301
51	CS-delta5D-THRSP	Thraustochytrium sp.	1320	23302-24621	delta5 desaturase gene; Qiu, X. et al. (2001). Journal of Biological Chemistry 276, 31561-31566	Altered fatty acids and oils
52	V-intervening-SYNTH	Synthetic	21	24622-24642
53	t-setl-BRANA	Brassica napus	614	24643-25256	Terminator of SETL. Bauer, J. and Senger, T. (2010). Patent WO2010000708, WIPO
54	V-intervening-SYNTH	Synthetic	146	25257-25402
55	P-arc5a-PHAVU	Phaseolus vulgaris	1151	25403-26553	Promoter of arcelin; Goossens, A. et al. (1999). Plant Physiology 120, 1095-1104
56	V-intervening-SYNTH	Synthetic	10	26554-26563
57	CS-delta4D-THRSP	Thraustochytrium sp.	1560	26564-28123	delta4 desaturase gene; Qiu, X. et al. (2001). Journal of Biological Chemistry 276, 31561-31566	Altered fatty acids and oils
58	V-intervening-SYNTH	Synthetic	13	28124-28136
59	T-arc5a-PHAVU	Phaseolus vulgaris	600	28137-28736	Terminator of arcelin; Goossens, A. et al. (1999). Plant Physiology 120, 1095-1104
60	V-intervening-SYNTH	Synthetic	92	28737-28828
61	P-pxr-LINUS	Linum usitatissimum	1727	28829-30555	Promoter of peroxiredoxin. Duwenig, E. and Loyall, L.P. (2006). Patent WO2006089950 A2, WIPO		sequence contains a cytosine to adenine nucleotide change; this nucleotide change does not result in an amino acid substitution.
62	I-AGO4	Arabidopsis thaliana	758	30556-31313	Intron-containing 5' UTR of gene AGO4; Zilberman, D. et al. (2003). Science 299, 716-719
63	V-intervening-SYNTH	Synthetic	15	31314-31328
64	CS-omega3D-PYTIR	Pythium irregulare	1092	31329-32420	omega3 desaturase gene; Cheng, B. et al. (2010). Transgenic Research 19, 221-229	Altered fatty acids and oils
65	V-intervening-SYNTH	Synthetic	56	32421-32476
66	T-pxr-ARATH	Arabidopsis thaliana	400	32477-32876	Terminator of peroxidin. Haslekås, C. et al. (1998). Plant Molecular Biology 36, 833-845
67	V-intervening-SYNTH	Synthetic	135	32877-33011
68	P-cnl1-LINUS	Linum usitatissimum	1064	33012-34075	Promoter of the conlinin1 gene. Truska et al. (2003). Plant Phys and Biochem, 41,141-147
69	I-At1g65090	Arabidopsis thaliana	455	34076-34530	Intron-containing 5' UTR of locus At1g65090; Braybrook, S.A. et al. (2006). PNAS 103, 3468-3473
70	V-intervening-SYNTH	Synthetic	9	34531-34539
71	CS-delta4D-DIALT	Diacronema lutheri	1338	34540-35877	delta4 desaturase gene from Diacronema Lutheri (also known as Pavlova lutheri); Tonon, T. et al. (2003). FEBS Letters 553, 440-444	Altered fatty acids and oils
72	V-intervening-SYNTH	Synthetic	21	35878-35898
73	T-ocs-RHIRD	Agrobacterium tumefaciens	192	35899-36090	3' untranslated region (3' UTR) of the octopine synthase gene from Agrobacterium tumefaciens Ti-Plasmid. DeGreve et al. (1982) J Mol Appl Genet. 1(6):499-511
74	V-intervening-SYNTH	Synthetic	193	36091-36283
75	P-fae1-BRANA	Brassica napus	1430	36284-37713	Promoter of fatty acid elongase. Han, J. et al. (2001). Plant Molecular Biology 46, 229-239
76	I-At1g62290	Arabidopsis thaliana	847	37714-38560	Intron-containing 5' UTR of locus At1g62290 (aspartyl protease family protein); Chen, X. et al. (2002). Eur J Biochem 269, 4675-4684
77	V-intervening-SYNTH	Synthetic	7	38561-38567
78	CS-delta5E-OSTTA	Ostreococcus tauri	903	38568-39470	delta5 elongase gene; Zank, T. et al. (2005). Patent WO2005012316 A2, WIPO	Altered fatty acids and oils
79	V-intervening-SYNTH	Synthetic	16	39471-39486
80	T-fae1-ARATH	Arabidopsis thaliana	400	39487-39886	Terminator of fatty acid elongase. Rossak, M. et al. (2001). Plant Molecular Biology 46, 717-725
82	V-intervening-SYNTH	Synthetic	118	39887-40004
84	P-ubi4-PETCR	Petroselinum crispum	394	40005-40398	Promoter of ubiquitin. Kawalleck, P. et al. (1993). Plant Molecular Biology 21, 673-684
86	I-ubi4-PETCR	Petroselinum crispum	588	40399-40986	Intron of ubiquitin; Kawalleck, P. et al. (1993). Plant Molecular Biology 21, 673-684
87	V-intervening-SYNTH	Synthetic	7	40987-40993
89	CS-ahas-ARATH	Arabidopsis thaliana	2013	40994-43006	Acetohydroxy acid synthase gene (mutated) from Arabidopsis thaliana. Chang and Duggleby (1998). Biochem J, 333, 765-777	ALS/AHAS inhibitor tolerance
91	T-ahas-ARATH	Arabidopsis thaliana	780	43007-43786	3' untranslated region (3' UTR) of the acetohydroxy acid synthase gene. Mazur et al. (1987) Plant Physiol, 85(4): 1110-1117
92	V-intervening-SYNTH	Synthetic	88	43787-43874
93	V-LB-RHIRD	Agrobacterium tumefaciens	136	43875-44010			truncated

Related DNA Sequences:

Acc.No. Reference Sequence Size (bp) Annotation

USDA-APHIS No 43818

Acc.No.	Reference	Sequence	Size (bp)	Annotation
	USDA-APHIS	No	43818

Name:	insert2_LBFLFK
Comment:	insert2_LBFLFK is identical to insert1_LBFLFK except for the nucleotide changes and rearrangement/truncations of or the RB and LB. insert2_LBFLFK (43 779 bp) has a 184 bp truncation of the 5' end of the RB and a 53 bp truncation of the 3' of the LB. Lastly, there was a guanine to thymine nucleotide change in insert2_LBFLFK that is in the coding sequence of the delta-4 desaturase gene, CS-delta4D-DIALT. This change resulted in an alanine to serine amino acid substitution (A102S) in the delta4D protein. The amino acid change have no impact on the function or activity of the protein.
Map:

Genetic Elements:

Related DNA Sequences:

Detection Methods

13 results

Name	Type	Target	Description	Validation	Standardisation	Verification
QL-ELE-T-OCS-F/T-OCS-R	element-specific	T-ocs-RHIRD	Qualitative real-time PCR (TaqMan) method for detection of octopine synthase terminator of Agrobacterium tumefaciens (Debode et al., 2013)	in-house validation	none	1
QL-ELE-T-35S-F/T-35S-R	element-specific	T-Cauliflower mosaic virus	Qualitative real-time PCR (TaqMan) method for detection of the 35S terminator from Cauliflower mosaic virus (Debode et al., 2013)	in-house validation	none	1
QL-ELE-00-023	element-specific	T-Cauliflower mosaic virus	Qualitative real-time PCR (SYBRGreen) method for detection of T-35S pCAMBIA sequences (EU-RL GMFF GMOMETHODS database as of January 20, 2017).	ring trial validation	EU reference method	2
QL-ELE-00-024	element-specific	T-rbcS_E9-PEA	Qualitative real-time PCR (TaqMan) method for detection of the terminator of the pea ribulose-1,5-bisphosphate carboxylase small subunit (rbcS) E9 gene (EU-RL GMFF GMOMETHODS database as of January 20, 2017).	ring trial validation	EU reference method	2
QL-TAX-BN-003	taxon-specific	Brassica napus	Qualitative real-time PCR (SYBRGreen) method for detection of oilseed rape cruciferin A gene(Barbau-Piednoir et al., 2014)	ring trial validation	unknown	1
QL-TAX-BN-004	taxon-specific	Brassica napus	Qualitative conventional PCR method for detection of oilseed rape high mobility group protein I/Y gene (Pan et al., 2007).	ring trial validation	unknown	1
QT-EVE-BN-012	event-specific	LBFLFK	Quantitative real-time PCR (TaqMan) method for detection of oilseed rape event LBFLFK (EU-RL GMFF GMOMETHODS database as of October 12, 2022)	ring trial validation	EU reference method	1
QT-EVE-BN-013	event-specific	LBFLFK	Quantitative real-time PCR (TaqMan) method for detection of oilseed rape event LBFLFK locus2 (EU-RL GMFF GMOMETHODS database as of October 12, 2022)	ring trial validation	EU reference method	1
QT-TAX-BN-001	taxon-specific	Brassica napus	Quantitative real-time PCR (TaqMan) method for detection of oilseed rape acyl-ACP thioesterase (FatA) gene (EU-RL GMFF GMOMETHODS database as of January 23, 2014) Estimated number of target copies per haploid genome (Jacchia et al. 2018): 1	ring trial validation	EU reference method	1
QT-TAX-BN-002	taxon-specific	Brassica napus	Quantitative real-time PCR (TaqMan) method for detection of oilseed rape cruciferin storage protein (EU-RL GMFF GMOMETHODS database as of January 27, 2014). Estimated number of target copies per haploid genome (Jacchia et al. 2018): 4	ring trial validation	EU reference method	1
QT-TAX-BN-003	taxon-specific	Brassica napus	Quantitative real-time PCR (TaqMan) method for detection of oilseed rape cruciferin A gene (Jacchia et al. 2019) (EU-RL GMFF GMOMETHODS database as of January 15, 2020)	ring trial validation	EU reference method	1
QT-TAX-BN-012	taxon-specific	Brassica napus	Quantitative real-time PCR (TaqMan) method for detection of oilseed rape cruciferin A (CruA) gene (EU-RL GMFF GMOMETHODS database as of March 06, 2014). Estimated number of target copies per haploid genome (Jacchia et al. 2018): 2	ring trial validation	EU reference method	1
QT-TAX-FatA primer1/FatA primer2	taxon-specific	Brassica napus	Quantitative real-time PCR (TaqMan) method for the detection of oilseed rape (Monsanto, 2004, A recommended procedure for real-time quantitative TaqMan PCR for Roundup Ready canola RT73). Estimated number of target copies per haploid genome (Jacchia et al. 2018): 2	in-house validation	none	1

Catalogue number	Source	Value	Type	Certified	Available
AOCS 0117-A	AOCS	<0.1 %	dried powder	yes	yes
AOCS 0117-E	AOCS	>99.1 %	dried powder	yes	yes

European GMO Initiative for a Unified Database System

GMO detailed information

GMO:

Alias:

UID:

Tradename:

Companies/Institutions:

Species:

Traits:

EU authorisation:

General Information

Links regarding to this GMO:

Inserts / Modification

Transformation Vectors

Detection Methods

Reference Materials

Authorisation

Literature reference