COMPOSITIONS AND METHODS COMPRISING PLANTS WITH REDUCED RAFFINOSE FAMILY OLIGOSACCHARIDES AND/OR HIGH PROTEIN CONTENT
Provided herein are plants, plant parts, a population of plants or plant parts, and plant products (e.g., seed composition, protein and/or oil composition) comprising reduced galactinol synthase (GAS) and/or raffinose synthase (RS) activity, and compositions and methods of producing such plants and plant parts. The plants, plant parts, population of plants or plant parts, or plant products can have a genetic mutation that reduces the GAS and/or RS activity, decreased RFO (e.g., raffinose, stachyose) content, increased sucrose content, and/or increased protein content. The mutation can be located at least partially in a GAS and/or RS gene or its homolog or its regulatory region.
1 . A plant or plant part comprising decreased galactinol synthase (GAS) activity and/or raffinose synthase (RS) activity compared to a control plant or plant part, wherein said plant or plant part comprises a genetic mutation that decreases the GAS activity and/or the RS activity.
2 . The plant or plant part of claim 1 , comprising decreased raffinose family oligosaccharide (RFO), raffinose, and/or stachyose content and/or increased sucrose content as compared to a control plant or plant part.
3 . (canceled)
4 . The plant or plant part of claim 1 , wherein the mutation comprises one or more insertions, substitutions, or deletions in at least one native GAS and/or RS gene or homolog thereof or regulatory region thereof in said plant or plant part, wherein:
an expression level of said at least one mutated GAS and/or RS gene or homolog thereof is reduced compared to corresponding at least one native GAS and/or RS gene or homolog thereof without said mutation; and/or
level or activity of a GAS and/or RS protein encoded by said at least one mutated GAS and/or RS gene or homolog thereof is reduced compared to a GAS and/or RS protein encoded by corresponding at least one native GAS and/or RS gene or homolog thereof without said mutation.
5 .- 6 . (canceled)
7 . The plant or plant part of claim 4 , comprising:
(i) a mutated Pisum sativum GAS gene comprising a G to A substitution of nucleotide 128 of SEQ ID NO: 5 or 25, or comprising a nucleic acid sequence of SEQ ID NO: 15 or 26;
(ii) a mutated Pisum sativum GAS gene comprising a deletion of nucleotides 115-119 of SEQ ID NO: 25, or comprising a nucleic acid sequence of SEQ ID NO: 27;
(iii) a mutated Pisum sativum GAS gene comprising a deletion of nucleotides 111-117 of SEQ ID NO: 25, or comprising a nucleic acid sequence of SEQ ID NO: 28;
(iv) a mutated Pisum sativum RS gene comprising a G to A substitution of nucleotide 1044 of SEQ ID NO: 7 or 31, or comprising a nucleic acid sequence of SEQ ID NO: 16 or 32;
(v) a mutated Pisum sativum RS gene comprising a deletion of nucleotides 1246-1253 of SEQ ID NO: 31, or comprising a nucleic acid sequence of SEQ ID NO: 33;
(vi) a mutated Pisum sativum RS gene comprising a deletion of nucleotides 1248-1254 of SEQ ID NO: 31, or comprising a nucleic acid sequence of SEQ ID NO: 34;
(vii) a mutated Pisum sativum GAS protein comprising a G to D substitution of amino acid 43 of SEQ ID NO: 9, or comprising an amino acid sequence of SEQ ID NO: 17;
(viii) a mutated Pisum sativum GAS protein encoded by the nucleic acid sequence of SEQ ID NO: 27, or comprising an amino acid sequence of SEQ ID NO: 29;
(ix) a mutated Pisum sativum GAS protein encoded by the nucleic acid sequence of SEQ ID NO: 28, or comprising an amino acid sequence of SEQ ID NO: 30;
(x) a truncated Pisum sativum RS protein comprising a deletion of amino acids 348-798 of SEQ ID NO: 11, or comprising an amino acid sequence of SEQ ID NO: 18;
(xi) a mutated Pisum sativum RS protein encoded by the nucleic acid sequence of SEQ ID NO: 33, or comprising an amino acid sequence of SEQ ID NO: 35;
(xii) a mutated Pisum sativum RS protein encoded by the nucleic acid sequence of SEQ ID NO: 34, or comprising an amino acid sequence of SEQ ID NO: 36;
(xiii) a mutated Glycine max GAS gene comprising a deletion of nucleotides 197-209 of SEQ ID NO: 42, or comprising a nucleic acid sequence of SEQ ID NO: 44;
(xiv) a mutated Glycine max GAS gene comprising a deletion of nucleotides 199-209 of SEQ ID NO: 42, or comprising a nucleic acid sequence of SEQ ID NO: 45;
(xv) a mutated Glycine max GAS gene comprising a deletion of nucleotide 204 of SEQ ID NO: 42, or comprising a nucleic acid sequence of SEQ ID NO: 46; and/or
(xvi) a mutated Glycine max GAS protein encoded by the nucleic acid sequence of any one of SEQ ID NOs: 44-46, or comprising an amino acid sequence of any one of SEQ ID NOs:
47-49.
8 .- 14 . (canceled)
15 . A population of plants or plant parts comprising the plant or plant part of claim 1 , wherein the population comprises decreased GAS and/or RS activity, decreased raffinose family oligonucleotide (RFO) content and/or increased sucrose content compared to a control population.
16 . (canceled)
17 . The plant or plant part of claim 1 , or a population of plants or plant parts comprising the plant or plant part of claim 1 , wherein the plant, plant part, or population of plants or plant parts comprises
seed raffinose content that is 85% or less relative to a control, seed stachyose content that is 50% or less relative to a control, and/or seed sucrose content that is increased by about 10% or more relative to a control; and/or
seed raffinose content of about 0.7% or less or about 0.3% or less, seed stachyose content of about 1.5% or less or about 1.4% or less, and/or seed sucrose content of about 3.0% or more or 3.7% or more dry weight of total seed content.
18 .- 20 . (canceled)
21 . A method for decreasing raffinose family oligosaccharide (RFO) content and/or increasing protein content in a plant or plant part, said method comprising decreasing galactinol synthase (GAS) activity and/or raffinose synthase (RS) activity in the plant or plant part.
22 . The method of claim 21 , comprising introducing a genetic mutation that decreases the GAS and/or RS activity into said plant or plant part, wherein:
the GAS and/or RS activity is decreased; and
RFO, raffinose, and/or stachyose content is decreased and/or sucrose content is increased in the plant or plant part relative to a control plant or plant part.
23 .- 26 . (canceled)
27 . The method of claim 22 , wherein:
(i) the mutation comprises a G to A substitution of nucleotide 128 of SEQ ID NO: 5 or 25, or said plant or plant part comprises a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 15 or 26 when said mutation is introduced;
(ii) the mutation comprises a deletion of nucleotides 115-119 of SEQ ID NO: 25, or said plant or plant part comprises a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 27 when said mutation is introduced;
(iii) the mutation comprises a deletion of nucleotides 111-117 of SEQ ID NO: 25, or said plant or plant part comprises a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 28 when said mutation is introduced;
(iv) the mutation comprises a G to A substitution of nucleotide 1044 of SEQ ID NO: 7 or 31, or said plant or plant part comprises a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 16 or 32 when said mutation is introduced;
(v) the mutation comprises a deletion of nucleotides 1246-1253 of SEQ ID NO: 31, or said plant or plant part comprises a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 33 when said mutation is introduced;
(vi) the mutation comprises a deletion of nucleotides 1248-1254 of SEQ ID NO: 31, or said plant or plant part comprises a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 34 when said mutation is introduced;
(vii) the mutation produces a G to D substitution of amino acid 43 of SEQ ID NO: 9, or said plant or plant part comprises a polypeptide comprising an amino acid sequence of SEQ ID NO: 17 when said mutation is introduced;
(viii) the mutation produces a mutated Pisum sativum GAS protein comprising an amino acid sequence of SEQ ID NO: 29 or 30;
(ix) the mutation produces a deletion of amino acids 348-798 of SEQ ID NO: 11, or said plant or plant part comprises a polypeptide comprising an amino acid sequence of SEQ ID NO:
18 when said mutation is introduced;
(x) the mutation produces a mutated Pisum sativum RS protein comprising an amino acid sequence of SEQ ID NO: 35 or 36;
(xi) the mutation comprises a deletion of nucleotides 197-209 of SEQ ID NO: 42, or said plant or plant part comprises a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 44 when said mutation is introduced;
(xii) the mutation comprises a deletion of nucleotides 199-209 of SEQ ID NO: 42, or said plant or plant part comprises a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 45 when said mutation is introduced;
(xiii) the mutation comprises a deletion of nucleotide 204 of SEQ ID NO: 42, or said plant or plant part comprises a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 46 when said mutation is introduced; and/or
(xiv) the mutation produces a mutated Glycine max GAS protein comprising an amino acid sequence of any one of SEQ ID NO: 47-49.
28 . (canceled)
29 . The method of claim 22 , further comprising contacting the plant or plant part with a mutagen, thereby introducing said mutation into said plant or plant part.
30 .
31 . The method of claim 22 , further comprising introducing editing reagents or a nucleic acid construct encoding said editing reagents into said plant, plant part, or plant cell, thereby introducing said mutation into said plant or plant part.
32 . The method of claim 31 , wherein said editing reagents comprise at least one nuclease and/or one or more guide RNAs (gRNAs),
wherein the nuclease cleaves a target site in a genome of said plant, plant part, or plant cell, and wherein said mutation is introduced at said cleaved target site, and
wherein the one or more gRNAs comprise a nucleic acid sequence complementary to a region of a nucleic acid sequence comprising at least one native GAS or RS gene or homolog thereof or regulatory region thereof in said plant or plant part.
33 .- 37 . (canceled)
38 . The method of claim 32 , wherein at least one of the one or more gRNAs comprises a nucleic acid sequence encoded by:
(i) a nucleic acid sequence that shares at least 80% sequence identity with a nucleic acid sequence of SEQ ID NO: 13, 14, or 52; or
(ii) the nucleic acid sequence of SEQ ID NO: 13, 14, or 52.
39 .- 42 . (canceled)
43 . A plant or plant part produced by the method of claim 21 , wherein said plant or plant part comprises reduced GAS and/or RS activity and/or decreased RFO, raffinose, and/or stachyose content and/or increased sucrose content compared to a control plant or plant part compared to a control plant or plant part.
44 .- 51 . (canceled)
52 . A composition produced from the plant or plant part of claim 1 or a population of plants or plant parts comprising the plant or plant part of claim 1 , wherein the composition is a seed composition, a protein composition, an oil composition, and/or a food or beverage product.
53 - 55 . (canceled)
56 . A pea composition comprising raffinose content of about 0.7% or less or 0.3% or less, stachyose content of about 1.5% or less or 1.4% or less dry weight, and/or sucrose content of about 3.0% or more or 3.7% or more dry weight.
57 .- 62 . (canceled)
63 . The pea composition of claim 6256 , comprising:
(i) a mutated Pisum sativum GAS gene comprising a G to A substitution of nucleotide 128 of SEQ ID NO: 5 or 25, or comprising a nucleic acid sequence of SEQ ID NO: 15 or 26;
(ii) a mutated Pisum sativum GAS gene comprising a deletion of nucleotides 115-119 of SEQ ID NO: 25, or comprising a nucleic acid sequence of SEQ ID NO: 27;
(iii) a mutated Pisum sativum GAS gene comprising a deletion of nucleotides 111-117 of SEQ ID NO: 25, or comprising a nucleic acid sequence of SEQ ID NO: 28;
(iv) a mutated Pisum sativum RS gene comprising a G to A substitution of nucleotide 1044 of SEQ ID NO: 7 or 31, or comprising a nucleic acid sequence of SEQ ID NO: 16 or 32;
(v) a mutated Pisum sativum RS gene comprising a deletion of nucleotides 1246-1253 of SEQ ID NO: 31, or comprising a nucleic acid sequence of SEQ ID NO: 33;
(vi) a mutated Pisum sativum RS gene comprising a deletion of nucleotides 1248-1254 of SEQ ID NO: 31, or comprising a nucleic acid sequence of SEQ ID NO: 34;
(vii) a mutated Pisum sativum GAS protein comprising a G to D substitution of amino acid 43 of SEQ ID NO: 9, or comprising an amino acid sequence of SEQ ID NO: 17;
(viii) a mutated Pisum sativum GAS protein encoded by the nucleic acid sequence of SEQ ID NO: 27, or comprising an amino acid sequence of SEQ ID NO: 29;
(ix) a mutated Pisum sativum GAS protein encoded by the nucleic acid sequence of SEQ ID NO: 28, or comprising an amino acid sequence of SEQ ID NO: 30;
(x) a truncated Pisum sativum RS protein comprising a deletion of amino acids 348-798 of SEQ ID NO: 11, or comprising an amino acid sequence of SEQ ID NO: 18;
(xi) a mutated Pisum sativum RS protein encoded by the nucleic acid sequence of SEQ ID NO: 33, or comprising an amino acid sequence of SEQ ID NO: 35; and/or
(xii) a mutated Pisum sativum RS protein encoded by the nucleic acid sequence of SEQ ID NO: 34, or comprising an amino acid sequence of SEQ ID NO: 36.
64 . (canceled)
65 . A nucleic acid molecule comprising a nucleic acid sequence of a mutated GAS or RS gene or coding sequence thereof wherein said nucleic acid sequence:
(i) has at least 80% identity to a nucleic acid sequence of any one of SEQ ID NOs: 15, 16, 26-28, 32-34, and 44-46;
(ii) comprises the nucleic acid sequence of any one of SEQ ID NOs: 15, 16, 26-28, 32-34, and 44-46;
(iii) encodes a polypeptide comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence of any one of SEQ ID NOs: 17, 18, 29, 30, 35, 36, and 47-49; and/or
(iv) encodes a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 17, 18, 29, 30, 35, 36, and 47-49.
66 . A DNA construct comprising, in operable linkage:
(i) a promoter that is functional in a plant cell; and
(ii) the nucleic acid molecule of claim 65 .
67 . A cell comprising the nucleic acid molecule of claim 65 , or the DNA construct comprising, in operable linkage, a promoter that is functional in a plant cell and the nucleic acid molecule of claim 65 .
68 . (canceled)