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1. WO2007107807 - PRODUCTION D'ACIDE ALPHA-LINOLÉNIQUE DANS DES TOURNESOLS

Note: Texte fondé sur des processus automatiques de reconnaissance optique de caractères. Seule la version PDF a une valeur juridique

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We Claim,

I) A method of transformation of Helianthus annuus with Agrobacterium tumefaciens harboring the recombinant vector comprising the nucleic acid sequence encoding the enzymes required for the alpha-linolenic acid biosynthesis, comprising of:

a) Construction of the recombinant vector incorporating the nucleic acid sequence encoding the enzyme delta- 15 desaturase.
b) Agrobacterium mediated gene transfer of the vector of step a) into the plant cells or plant tissues of Helianthus annuus under conditions optimal for infection. c) A step of selection of the putative transformants from the population of
genetically non-transformed plant cells/tissues on a selection medium.
d) A step of regeneration of the selected plant cells or plant tissues of the host plant of step b)

2) A method of obtaining the nucleic acid fragment encoding the delta- 15 desaturase enzyme comprising:

a) designing primers for amplification of the open reading frame of delta- 15
desaturase gene in Brassica juncea.
b) Amplification of cDNA of Brassica juncea with the designed primers of step a) c) The amplified nucleic acid sequence wherein at least one of the nucleotide
sequence is modified in that the expression of the thus modified DNA occurs in the host plant.
d) The nucleic acid sequence encoding delta- 15 desaturase and having a nucleic acid sequence selection from the group comprising SEQ IDl and the complementary strand thereof.

3) A method according to step a) of claim 1, wherein the sequence represented in SEQ ID 1 is introduced into vector pC AMBIA 1390 operably linked to a ubiquitin promoter.

4) The vector construct comprising the isolated nucleic acid sequence of SEQ IDl operably linked to the ubiquitin promoter.

5) The vector construct of claim 4 comprising a selectable marker gene.

6) The vector construct of claim 4, wherein the selectable marker gene is xylose isomerase.

7) A method according to any of the preceding claims, wherein the corresponding amino acid sequence of the expressed protein is represented in SEQ ID 1.

8) A method according to claim 1, wherein the host plant species used is Helianthus annuus.

9) A method according to claiml, wherein the step of transformation includes a stage-wise co-cultivation of explants is used which comprises:

a) A step of preparing an explant by excision of the split embryonic axis to remove the cotelydons, radicula, and the leaf primordia in order to expose the meristem. b) A step of culturing the microorganisms belonging to the genus Agrobacterium tumefaciens comprising the nucleic acid sequences required for Alpha-linolenic acid production.
c) A step of infection and the co-cultivation of the said explants with the
Agrobacterium containing culture medium of step b) under vacuum at 200 atm pressure for 15 minutes and the subsequent transfer into the co-cultivation
medium 2 days at 26 ° C under dark.
d) A step of transferring the explants into the selection and the regeneration medium containing a selection agent cultured under photo period of 16 hours light and 8 hours dark condition for 2-3 hours.
e) A step of subsequent transfer of the selected explants into the elongation and the shoot induction medium.

f) A step further comprising the regeneration of the selected transformed shoots into the host plant of claim 4.

10) A method according to the preceding claim, characterized in that the confirmation of transformation of the said selectable marker gene in the regenerated plantlets is done using the known molecular techniques of screening.

11) A method of production of alpha-linolenic acid by providing the host plant cell comprising
a) The nucleic acid sequence encoding delta- 15 desaturase polypeptide.
b) Culturing the host plant cells under conditions wherein the nucleic acid sequence encoding the enzyme delta- 15 desaturase is expressed and linoleic acid is
converted to alpha-linolenic acid
c) Optionally recovering the alpha-linolenic acid of step b)

12) The culture of the plant cells produced by the method according to any of the said preceding claims wherein the cells are enriched with the polyunsaturated fatty acid alpha-linolenic acid.

13) A plant that has been regenerated from the culture of the plant cells of claim 11

14) A transgenic plant comprising the isolated nucleic acid of claim 2) wherein the said nucleic acid sequence is integrated into the genome of the said plant by Agrobacterium mediated gene transfer.

15) The progeny of the transgenic plant of claim 14.

16) The seeds of the transgenic plant of Claim 14.