Oleic Acid-Enriched Plant Body Having Genetically Modified FAD2 And Production Method Thereof

Abstract

The present invention relates to an artificially manipulated unsaturated fatty acid biosynthesis-associated factor and use thereof to increase the content of a specific unsaturated fatty acid of a plant body. More particularly, the present invention relates to a system capable of artificially controlling unsaturated fatty acid biosynthesis and a plant body produced thereby, which include an artificially manipulated unsaturated fatty acid biosynthesis-associated factor to control unsaturated fatty acid biosynthesis and a composition capable of artificially manipulating the factor. In a specific aspect, the present invention relates to artificially manipulated unsaturated fatty acid biosynthesis-associated factors such as FAD2, FAD3, FADE, FAD7 and FAD8 and/or an unsaturated fatty acid biosynthesis controlling system by an expression product thereof.

1 - 25 . (canceled) 
     
     
         26 . A plant comprising an artificially manipulated genome, wherein the artificially manipulated genome includes one or more modifications selected from the group consisting of
 (i) a deletion of 1 to 30 nucleotides;   (ii) an insertion of one or more foreign nucleotides;   (iii) a deletion of 1 to 30 nucleotides and an insertion of one or more foreign nucleotides; and   (iv) a substitution with one or more nucleotides different from a wild-type gene, in a continuous 1 to 50 nucleotides sequence including a PAM sequence in a nucleic acid sequence constituting a FAD2 gene,   wherein the PAM sequence is one or more sequences selected from 5′-NGG-3′; 5′-NNNNRYAC-3′; 5′-NNAGAAW-3′; 5′-NNNNGATT-3′; 5′-NNGRR(T)-3′; and 5′-TTN-3′, wherein the each N is independently A, T, C or G, the each R is independently A or G, the Y is C or T, and the W is A or T,   wherein the plant comprising an artificially manipulated genome has at least one of the phenotype selected from the group consisting of, as compared with a wild type plant:   (a) a decreased expression of a FAD2 protein;   (b) a decreased RNA transcripts of a FAD2 gene;   (c) an increased expression of a mutated FAD2 protein;   (d) an increased content of C8˜24:D1 unsaturated fatty acid; and   (e) a decreased content of C8˜24:D2 unsaturated fatty acid.   
     
     
         27 . The plant of  claim 26 , wherein the continuous 1 to 50 nucleotides sequence including a PAM sequence in a nucleic acid sequence constituting a FAD2 gene comprises at least one of the nucleotide sequence selected from SEQ ID NO: 1 to 30. 
     
     
         28 . The plant of  claim 26 , wherein the C8˜24:D1 unsaturated fatty acid is a C16˜22:D1 unsaturated fatty acid. 
     
     
         29 . The plant of  claim 26 , wherein the C8˜24:D1 unsaturated fatty acid is a C18:D1 unsaturated fatty acid. 
     
     
         30 . The plant of  claim 26 , wherein the C8˜24:D2 unsaturated fatty acid is a C16˜22:D2 unsaturated fatty acid. 
     
     
         31 . The plant of  claim 26 , wherein the C8˜24:D2 unsaturated fatty acid is a C18:D2 unsaturated fatty acid. 
     
     
         32 . The plant of  claim 26 , wherein the plant is a soybean. 
     
     
         33 . A composition for gene manipulation, which is used to produce a plant including an artificially manipulated genome, comprising:
 a guide nucleic acid capable of targeting a FAD2 gene, or a nucleic acid sequence encoding the same; and   an editor protein, or a nucleic acid sequence encoding the same,   wherein the guide nucleic acid is a RNA sequence capable of binding a target region in a nucleic acid sequence constituting a FAD2 gene,   wherein the target region gene comprises at least one of the nucleotide sequence selected from SEQ ID NO: 1 to 30, and   wherein the editor protein includes one or more proteins selected from the group consisting of a  Streptococcus pyogenes -derived Cas9 protein, a  Campylobacter jejuni -derived Cas9 protein, a  Streptococcus thermophilus -derived Cas9 protein, a  Staphylococcus aureus -derived Cas9 protein, a  Neisseria meningitidis -derived Cas9 protein, and a Cpf1 protein.   
     
     
         34 . The composition of  claim 33 , wherein the composition for gene manipulation is formed in an  agrobacterium  vector system. 
     
     
         35 . The composition of  claim 33 , wherein the composition for gene manipulation is formed in a viral vector system. 
     
     
         36 . The composition of  claim 35 , wherein the viral vector includes one or more selected from a mosaic virus, a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus (AAV), a vaccinia virus, a poxvirus and a herpes simplex virus. 
     
     
         37 . A method for producing a plant including an artificially manipulated genome, comprising an introducing (administering) a composition to a subject,
 wherein the subject is a plant cell, a seed, a part of a plant body or whole plant body,   wherein the composition comprising:   a guide nucleic acid capable of targeting a FAD2 gene, or a nucleic acid sequence encoding the same; and   an editor protein, or a nucleic acid sequence encoding the same,   wherein the guide nucleic acid is a RNA sequence capable of binding a target region in a nucleic acid sequence constituting a FAD2 gene,   wherein the target region gene comprises at least one of the nucleotide sequence selected from SEQ ID NO: 1 to 30,   wherein the editor protein includes one or more proteins selected from the group consisting of a  Streptococcus pyogenes -derived Cas9 protein, a  Campylobacter jejuni -derived Cas9 protein, a  Streptococcus thermophilus -derived Cas9 protein, a  Staphylococcus aureus -derived Cas9 protein, a  Neisseria meningitidis -derived Cas9 protein, and a Cpf1 protein,   wherein the artificially manipulated genome includes one or more modifications selected from the group consisting of   (i) a deletion of 1 to 30 nucleotides;   (ii) an insertion of one or more foreign nucleotides;   (iii) a deletion of 1 to 30 nucleotides and an insertion of one or more foreign nucleotides; and   (iv) a substitution with one or more nucleotides different from a wild-type gene,   in a continuous 1 to 50 nucleotides sequence including a PAM sequence in a nucleic acid sequence constituting a FAD2 gene,   wherein the PAM sequence is one or more sequences selected from 5′-NGG-3′; 5′-NNNNRYAC-3′; 5′-NNAGAAW-3′; 5′-NNNNGATT-3′; 5′-NNGRR(T)-3′; and 5′-TTN-3′, wherein the each N is independently A, T, C or G, the each R is independently A or G, the Y is C or T, and the W is A or T,   wherein the plant comprising an artificially manipulated genome has at least one of the phenotype selected from the group consisting of, as compared with a wild type plant:   (a) a decreased expression of a FAD2 protein;   (b) a decreased RNA transcripts of a FAD2 gene;   (c) an increased expression of a mutated FAD2 protein;   (d) an increased content of C8˜24:D1 unsaturated fatty acid; and   (e) a decreased content of C8˜24:D2 unsaturated fatty acid.   
     
     
         38 . The method of  claim 37 , wherein the composition is formed in an  agrobacterium  vector system. 
     
     
         39 . The method of  claim 37 , wherein the composition is formed in a viral vector system. 
     
     
         40 . The method of  claim 39 , wherein the viral vector includes one or more selected from a mosaic virus, a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus (AAV), a vaccinia virus, a poxvirus and a herpes simplex virus.