Leghemoglobin in soybean

Abstract

Soybean plants producing soybean seeds comprising leghemoglobin are produced by modifying the genome of the soybean plant. Soybean plants, soybean seeds and soy protein compositions comprising leghemoglobin are provided. Soybean plants, soybean seeds and soy protein compositions comprising leghemoglobin and additionally one or more of high oleic acid, low linolenic acid, high protein, low stachyose, low raffinose and low protease inhibitors are provided. Protein compositions comprising leghemoglobin, such as soy isolates and concentrates can be made from the soybean seeds. Additionally, methods for generating and using plants, seeds and protein compositions comprising leghemoglobin are disclosed.

1 . A soybean seed comprising a leghemoglobin protein in an amount of at least 0.5% of total protein in the soybean seed, wherein the soybean seed does not contain a recombinant construct comprising a leghemoglobin coding sequence integrated into its genome. 
     
     
         2 . The soybean seed of  claim 1 , wherein the soybean seed genome has been modified to introduce an insertion, deletion or substitution into a native leghemoglobin gene. 
     
     
         3 . The soybean seed of  claim 1 , wherein the soybean seed genome has been modified to introduce (i) an insertion, wherein the insertion comprises a regulatory enhancer or promotor sequence or (ii) a substitution wherein the substitution creates or enhances a regulatory enhancer or promotor sequence. 
     
     
         4 . The soybean seed of  claim 1 , wherein the soybean seed genome has been modified to introduce an insertion, and the insertion comprises a targeting sequence operably linked to a leghemoglobin coding sequence of the leghemoglobin gene, the targeting sequence targeting the leghemoglobin to an intracellular compartment. 
     
     
         5 . The soybean seed of  claim 4 , wherein the targeting sequence targets the leghemoglobin to the plastid. 
     
     
         6 . The soybean seed of  claim 5 , wherein the targeting sequence comprises a polynucleotide encoding a polypeptide having at least 95% identity to SEQ ID NO: 32. 
     
     
         7 . The soybean seed of  claim 1 , wherein the soybean seed genome has been modified to replace all or part of a coding sequence of a seed storage protein with a leghemoglobin coding sequence. 
     
     
         8 . The soybean seed of  claim 7 , wherein the soybean seed genome has been modified to introduce an insertion, and the insertion comprises a targeting sequence operably linked to the leghemoglobin coding sequence which targets the leghemoglobin to an intracellular compartment. 
     
     
         9 . The soybean seed of  claim 8 , wherein the targeting sequence targets the leghemoglobin to the plastid. 
     
     
         10 . The soybean seed of  claim 9 , wherein the targeting sequence comprises a polynucleotide encoding a polypeptide having at least 95% identity to SEQ ID NO: 32. 
     
     
         11 .- 24 . (canceled) 
     
     
         25 . A soybean seed comprising a modified leghemoglobin gene, wherein the modified leghemoglobin gene comprises at least one deletion, insertion or substitution into the native leghemoglobin gene, and wherein the leghemoglobin protein is expressed in the soybean seed in an amount sufficient to impart a pink color to the soybean seeds in a transverse section or the seed. 
     
     
         26 . The soybean seed of  claim 25 , wherein the leghemoglobin protein is expressed in an amount of at least 0.5% of the total seed protein. 
     
     
         27 . The soybean seed of  claim 25 , wherein the modified leghemoglobin gene comprises an insertion of a promoter or a regulatory enhancing sequence. 
     
     
         28 . The soybean seed of  claim 25 , wherein the modified leghemoglobin gene comprises an insertion of a targeting sequence operably linked to a leghemoglobin coding sequence of the leghemoglobin gene, the targeting sequence targeting the leghemoglobin to an intracellular compartment. 
     
     
         29 . The soybean seed of  claim 28 , wherein the targeting sequence targets the leghemoglobin to the plastid. 
     
     
         30 . The soybean seed of  claim 29 , wherein the targeting sequence comprises a polynucleotide encoding a polypeptide having at least 95% identity to SEQ ID NO: 32. 
     
     
         31 . The soybean seed of  claim 25 , wherein the soybean seed further comprises a recombinant construct integrated into its genome, the recombinant construct comprising a leghemoglobin coding sequence. 
     
     
         32 . (canceled) 
     
     
         33 . (canceled) 
     
     
         34 . The soybean seed of  claim 25 , wherein the soybean seed further comprises an insertion of a leghemoglobin coding sequence into a native seed storage protein gene such that the leghemoglobin coding sequence replaces all or part of the native storage protein gene coding sequence. 
     
     
         35 . The soybean seed of  claim 25 , further comprising one or more of the following characteristics: (i) an oleic acid content of at least 50% of the total seed fatty acids; (ii) a linolenic acid content of less than 3% of the total seed fatty acids;
 (iii) a protein content of at least 37% of the total weight of the soybean measured at or adjusted to 13% moisture; (iv) a Kunitz Trypsin protease inhibitor activity of less than 5% of that of a control unmodified soybean; (v) a Bowman-Burke protease inhibitors activity of less than 5% of a control unmodified soybean; (vi) a stachyose content of less than 1% weight percent at 13% moisture; and (vii) a raffinose content of less than 0.5% weight percent at 13% moisture.   
     
     
         36 .- 73 . (canceled) 
     
     
         74 . A plant grown from the soybean seed of  claim 1 . 
     
     
         75 .- 79 . (canceled)