Access Rights to Background made available to the Parties: Partner 1 (slu)




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Access Rights to Background made available to the Parties:
Partner 1 (SLU).
Following FAR cDNA and gene constructs for E. coli expression and E. coli strains:

Jojoba FAR, At3g11980, At3g44560, At3g56700 long splicing version, At3g56700 short splicing version, At5g22500


Yeast strains with mutations in ARE1, ARE2, DGA1, LRO1 and combinations of them.

Partner 3 (Bayer)
Synthetic versions of the A. thaliana genes At3g44540 and At3g44550, codon optimized for expression in Brassicaceae
Synthetic versions of the mouse FAR1 gene (Genbank accession N°BC007178) and the mouse DGAT2 like wax synthase gene (GenBank accession N°AY611031) codon optimized for expression in Brassicaceae
[14C] octadec-12-enol

Partner 4 (Hubei)
C.abyssinica fad2 sequences: DQ518290,DQ518291,DQ518292
pCambia 2300-nap-Tmfae1 containing napin promoter and Tropaeolum fae1 gene
pCambia 2300-napin-LdLPAAT containing napin promoter and Limnanthes douglasii

LPAAT gene


Cambia 2300-napin-Bnfad2hairpin containing napin promoter and Brassica napus

fad2 hairpin (might be useful to silence Crambe fad2)


Crambe abyssinica materials with better tolerance to Alternaria
Interspecific hybrids and offsprings between C. abyssinica, C. kralikii and

C.hispanica


Partner 7 (RRes)
Synthetic genes derived from the amino acid sequences of the Trypanosoma brucei elongases ELO1, 2, 3 and 4.
Synthetic gene derived from the amino acid sequence of the Perkinsus marinus FAE1-like KCS  DQ508732
Synthetic gene derived from the amino acid sequence of the Eh86 viral ELO EhV077  YP_293831

Partner 8 (RWTHA)
Brassica napus FAE1 and Limnanthes douglaasii LPAAT constructs for seed specific expression.
Modified putative WS ORFs from Tetrahymena.


Partner 10 (CNRS)
CNRS-UTC : UMR CNRS 6022

Plasmids, genes availableas background for ICON :

  • Portion of gene encoding the -subunit of the Sinorhizobium meliloti propionyl-CoA carboxylase (PCCase), subcloned in vectors pTC322 (E. coli strain MT607) and pTC325 (E. coli strain DH5a) (Charles et Aneja, Gene, 1999, 226, 121-127.

  • Gene encoding the -subunit of the Sinorhizobium meliloti propionyl-CoA PCCase (REFLAX : QLK3-2000-00349).

  • Gene encoding the -subunit of the Sinorhizobium meliloti propionyl-CoA PCCase (REFLAX : QLK3-2000-00349).

  • Gene encoding the Acyl-Transferase (AT) domain of the Mycobacterium bovis BCG mycocerosic acid synthase (MAS) (plasmid pET 21b) : E. coli strain BL21 (DE3) (Mathur et Kollattukudy, J. Biol. Chem., 1992, 267, 19388-19395)

  • Gene encoding the Ketoacyl-Synthase (KS) domain of the Mycobacterium bovis BCG MAS (plasmid pET 21b): E. coli strain BL21 (DE3) (Mathur et Kollattukudy, J. Biol. Chem., 1992, 267, 19388-19395)

  • Gene encoding the threonin desaminase (TD) domain of the Escherichia coli (plasmid pQE 80): E. coli strain BL21 (DE3) (REFLAX : QLK3-2000-00349).

  • Gene sequence encoding the pea rbcS transit peptide, allowing chloroplastic expression (p35S-35S-PT- polyA: plasmid pJIT117): E. coli strain JM101 (Guérineau et al., Nucleic Acid Research, 1988, 16, 11380.

  • Gene sequence encoding the Arabidopsis thaliana transit peptide, allowing chloroplastic expression, (p35S-35S-PT-S65TmGFP4-3'nos) (Köhlner et al., Science, 1997, 276, 2039-2042)

  • pET-PCCBmx construct containing the ß-subunit of the Myxococcus xanthus propionyl-CoA carboxylase (REFLAX : QLK3-2000-00349).

  • pET-PCCA1 construct containing the -subunit of the Myxococcus xanthus propionyl-CoA carboxylase (REFLAX : QLK3-2000-00349).

  • pET-TD construct containing the TD of the Escherichia coli (REFLAX : QLK3-2000-00349).

  • pMAT construct containing the AT domain of the Mycobacterium bovis BCG (REFLAX : QLK3-2000-00349).

  • pKS construct containing the KS domain of the Mycobacterium bovis BCG (REFLAX : QLK3-2000-00349).

  • pET-KSe construct containing the KSe complete of KS domain of the Mycobacterium bovis BCG (REFLAX : QLK3-2000-00349).

  • pET-KSAT construct containing the complete KS-AT domain of the Mycobacterium bovis BCG (REFLAX : QLK3-2000-00349).

  • p35S SAM construct containing the SAM of the Arabidopsis thaliana (Boerjan et al. Plant Cell, 1994, 6, 1401-1414).

  • pbin-AT1 construct containing the AT domain of the Mycobacterium bovis BCG under control of promoter napin (REFLAX : QLK3-2000-00349).

  • pbin-KS3 construct containing the KS domain of the Mycobacterium bovis BCG under control of promoter napin (REFLAX : QLK3-2000-00349).

  • pbin-TD construct containing the TD of the Escherichia coli under control of promoter napin (REFLAX : QLK3-2000-00349).

  • pbin-PCCA4 construct containing the -subunit of the Myxococcus xanthus propionyl-CoA carboxylase under control of promoter napin (REFLAX : QLK3-2000-00349).

  • pCCBmx construct containing the ß-subunit of the Myxococcus xanthus propionyl-CoA carboxylase under control of promoter napin (REFLAX : QLK3-2000-00349).


Bacteria :

  • Escherichia coli wild types: FT1, ZK 126 and MG 1655 (Chang et Cronan, Mol. Microbiol. 1999, 33, 249-259)

  • Escherichia coli JC201 mutant in lpaat gene (Coleman, J Biol. Chem., 1990, 265, 17215-17221)

  • Modification des Escherichia coli mutant strains bearing the cfa synthase gene: YYC 1106, YYC 1272, YYC 1273 (Chang & Cronan, Mol. Microbiol. 1999, 33, 249-259).

  • Agrobacterium tumefaciens : C58C1 RifR : pGV2260 CbR à octopine (Deblaere et al., Nucl. Acids Res. 1985, 13, 4777-4788).

  • Agrobacterium tumefaciens : C58C1 RifR : GV3101 contenant le plasmide pGV2260 CbR à octopine (Deblaere et al., Nucl. Acids Res. 1985, 13, 4777-4788).

CNRS-Bordeaux : UMR CNRS 5200



Biological Material:
Genes:


  • Gene encoding the fatty acid reductase CER4 from Arabidopsis thaliana (At4g33790; Rowland et al., Plant Physiol. (2006) 142(3):866-77).

  • Gene encoding the fatty acid reductase FAR1 from Arabidopsis thaliana (At5g22500).

  • Gene encoding the fatty acid reductase FAR4 from Arabidopsis thaliana (At3g44540).

  • Gene encoding the fatty acid reductase FAR6 from Arabidopsis thaliana (At3g56700).

  • Gene encoding the fatty acid reductase PgFAR from Bombyx mori (Moto et al., Proc. Natl. Acad. Sci USA (2003) 100: 9156-9161).



Plasmids & Constructs:
The Arabidopsis genes mentioned above have been introduced in various vectors for expression in bacteria, yeast or plant using the GATEWAY® cloning technology (Invitrogen). All these constructs are summarized in the following table.





FAR3 (At4g33790)

FAR1 (At5g22500)

FAR4 (At3g44540)

FAR6 (At3g56700)

Promoter (pDONR221)

* (2kb)

* (2kb)

* (2kb)

* (2kb)

GUS Construct (pKGWFS7)

*

*

*

*

ORF (pDONR221)

* 1482pb

* 1476pb

* 1482pb

* 1584pb

ORF-Stop (pDONR221)

* 1479pb

* 1473pb

* 1479pb

* 1581pb

Nterm HA-tag (pDONR221)

* 1509pb

* 1503pb

* 1509pb

/

Yeast expression vector (pVT102U)

*

*

*

*

Bacteria expression (pDEST17)

*

*

/

*

Plant expression vector (pK7WGLD)

*

*

/

*

GFP Fusions (pK7WGF2 ou pK7FWG2)

*

*

/

*

RNAi (pK7GWIWG2)

*

*

/

*

Legend : (*) = obtained ; /  = in progress.
The Bombyx mori gene mentioned above is cloned in pESC-LEU as reported in the publication from Moto et al., (2003) as well as in the yeast expression vector pVT102U.

Bacteria :


  • Escherichia coli XL1 Blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F´ proAB lacIqZrM15 Tn10 (Tetr)] (Stratagen)

  • Escherichia coli DH5α F- s80lacZrM15 r(lacZYA-argF) U169 deoR recA1 endA1 hsdR17 (rk-, mk+) phoA supE44 t- thi-1 gyrA96 relA1 (Invitrogen)

  • Escherichia coli BL21 (DE3) F– ompT hsdSB (rB– mB–) gal dcm (DE3) Novagen




  • Agrobacterium tumefaciens C58C1 Contient le plasmide helper pMP90 (Koncz et al., 1992)

  • Agrobacterium tumefaciens GV3101 Contient le plasmide helper pMP90 (Koncz et al., 1992)



Yeast :

  • Saccharomyces cerevisiae strain BY4742 MATα, his3-Δ1, leu2-Δ0, lys2-Δ0, ura3-Δ0 (Brachmann et al., 1998).

  • Saccharomyces cerevisiae strain INVSC1 MATα, his3-Δ1, leu2, trp1-289, ura3-52 (Invitrogen)

  • Saccharomyces cerevisiae strain UTL7A MATα, ura3-52, leu2-3, 112, trp1

  • Saccharomyces cerevisiae strain C13ABYS86 (leu2, ura3, his, pra1, prb1, prc1, cps) (Bröker et al., 1991).


Plants :
Arabidopsis plants Col-0 transformed with the vectors mentioned in the previous table (35S, RNAi, GUS, GFP-fusion) have been generated and the selection of homozygous transgenic line is in progress.
Partner 11 (Warwick)
Arabidopsis jojoba-like lipase gene At5g04040
Partner 12
PRI-Germplasm of Crambe abyssinica, for example the existing cultivars Galactica and Nebula, but also if needed other germplasm already available at PRI as described in the breeding books of PRI
PRI-EMS mutant population based on Crambe abyssinica cv. 'Galactic' with over 10,000 M2-lines and the DNA-pools from those lines  and parental lines and offspring of these lines. 
DNA-sequences of glucosinolate genes  (especially targeted at  but not limited to epiprogoiterin) , FAD2  in Crambe abyssinica; dehiscence gene sequences from Arabidopsis in patent WO 2006/011795: "Method for preventing dehiscence and altering plant lignification" by Aharoni and Pereira to PRI.
 pMF binary vectors for DNA transfer containing  a  recombinase /LBD system  for  inducible excision of sequences between two recombination sites (Rs) together with a dual positive/negative selection system that can be used for introduction of new genes in the genome of plants and later inducing recombination in such a way that some of the new genes are removed again from the genome of  the  plant;  the pMF marker-free vector series can be used for example to use antibiotics resistance genes as  selectable marker genes and later , when desired,  remove the antiobiotics genes  and select for a homogeneously marker free GM-crop 

Partner 14 (CSIRO)


  1. Gene sequences for FAD2, FAD3 and FAE 1 subject to these conditions:

  1. Non exclusive use for ICON Project only.

  2. MTA conditions at Attachment 5 apply where relevant.




  1. RNAi, subject to these conditions:

  1. available non exclusively only as necessary to the Party undertaking silencing work within the ICON Project, for the duration of the genesilencing work.

  2. Field limited to use in Crambe.

  3. No sublicensing to third parties without CSIRO’s consent.



Publications require appropriate acknowledgement of CSIRO and contribution of RNAi to achieving results.
Partner 15 (CU)
Arabidopsis FAR genes.  The cDNAs of At3g44540, At3g44550, At5g22500, and At4g33790 (CER4) and gene constructs for expression in yeast


Partner 16 (DC)
Genes and gene constructs for plant seed expression constructs::
JojobaFAR+Jojoba WS
Jojoba FAR + Jojoba WS + Lunnaria annua FAE
AtFAR-At5g22500 + Jojoba WS

AtFAR-At3g11980 + Jojoba WS

WS from petunia
 
Transgenic plants:

 DsRed-Jojoba FAR + Jojoba WS in Arabidopsis/Nasturtium FAE background


DsRed-Jojoba WS + AtFAR-At5g22500 in Arabidopsis/Nasturtium FAE background

DsRed-AtFAR-At3g11980 + Jojoba WS in Arabidopsis/Nasturtium FAE background

DsRed-AtFAR-At5g22500 + Jojoba WS + in Arabidopsis high oleic acid background

DsRed-AtFAR-At3g11980 + Jojoba WS in Arabidopsis high palmitic acid background

Brassica juncea with jojoba FAR + WS

Jojoba WS + Jojoba FAR in Camelina sativa
Partner 18 (ISU)
cDNA clones for Arabidopsis wax synthase genes At5g55380, At5g55340, and At5g51420
Expression vector constructs for expressing cDNAs in E. coli, yeast, and plants
Vector constructs carrying promoter::GUS or Promoter::GFP transgenes for the Arabidopsis wax synthase genes At5g55380, At5g55340, and At5g51420
Partner 19 (NRC-PBI)
Easily-transformed breeding lines of B carinata (for example line C90-1163)

Crambe abyssinica FAE (genes and constructs)

Brassica carinata FAD2 RNAi (construct and expression lines)
B. carinata FAD2RNAi + Crambe FAE (transformed line)

Brassica carinata transformed with jojoba WS + jojoba FAR

Brassica carinata transformed with jojoba WS + jojoba FAR + Lunnaria annua FAE

Arabidopsis lpcat-ko and LPCAT-RNAi lines as host for WS + Jojoba FAR expression

cDNA clones encoding putative LPCAT candidates from Lesquerella fendleri (genes and constructs for expression in plants and yeast, transformed Arabidopsis lines) **when filing for IP completed.

cDNA encoding D12-oleate hydroxylase from Lesquerella lindheimeri  (genes and constructs for expression in plants and yeast, transformed Arabidopsis lines)

cDNA encoding Lesquerella fendleri ricinoleate condensing enzyme, KCS3 (genes and constructs for expression in plants and yeast, transformed Arabidopsis lines)

Arabidopsis lines transformed with a plant oleate D12 hydroxylase.


Partner 20 (UoS)
Putative WS and FAR genes from Euglena gracilis
Partner 21
cDNA library from germinating jojoba seeds. 4500 EST:s from the library
Partner 22 (USDA)
Jojoba FAR, jojoba WS and Lunaria annua FAE, all with codon optimised for Brassicacae.
Partner 23 (UBC)
Genes:

FAR: Arabidopsis CER4


Arabidopsis jojoba type WS cDNA : At3g51970, At1g34490, At1g34500, At5g55320, At5g55330, At5g55340, At5g55350, At5g55360, At5g55370, At5g55380, At1g34520, At5g51420

KCS: KCS45Lesquerella gene required for elongation of hydroxy fatty acids, CER6 FAE1 and FAE1 orthologs from B. napus, and L. annua.  Constructs for expression in yeast, epidermis specific expression (CER6) and seed-specific overexpression (LfKCS3, FAE1, and FAE1 orthologs). 



Mutants:

Arabidopsis fae1 mutant

Arabidopsis fae1/fad2 double mutant
Partner 24 (Uni Bonn)
WS/DGAT cDNA clones for: At3g49200, At5g22490, At5g53380, but we are not sure whether they are full-length or not.

 cDNAs were cloned into pTrcHis and transferred into E. coli: Ag3g49210, At5g12420, At5g16350, At5g37300,



 cDNAs were cloned into pYES2 and transferred into yeast: At3g49190, Ag3g49210, At5g12420, At5g37300,

  T-DNA insertion mutants. PCR shows that they are homozygous: At3g49190, At3g49200, Ag3g49210, At5g12420, At5g16350, At5g37300, At5g53390


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