Enhanced zein reduction in transgenic corn seed

Abstract

Anti-sense-oriented RNA gene suppression agents in the form of a loop of anti-sense-oriented RNA is produced in cells of transgenic organisms, e.g. plants, by transcription from a recombinant DNA construct that comprises in 5′ to 3′ order a promoter element operably linked to more than one anti-sense-oriented DNA element and one or more complementary DNA elements.

1 . A recombinant DNA construct for suppression of a plurality of maize zein target genes, comprising in 5′ to 3′ order a promoter element operably linked to a plurality of anti-sense-oriented DNA elements from a plurality of maize zein target genes, the anti-sense-oriented DNA elements designated in 5′ to 3′ order element 1, element 2, and a plurality of sense-oriented DNA elements from a plurality of maize zein target genes, designed in 5′ to 3′ order element 3, element 4, wherein the anti-sense-oriented DNA element 2 has at least a portion of its sequence at its 3′ end that is not complementary either to itself or to the sense-oriented DNA element 3, and wherein sense-oriented RNA transcribed by the sense-oriented DNA elements is complementary to the 5′-most end of anti-sense-oriented RNA transcribed by the anti-sense-oriented DNA elements.  
     
     
         2 . A recombinant DNA construct of  claim 1  wherein the anti-sense-oriented DNA elements 1 and 2 are from two different genes targeted for suppression and the sense-oriented DNA elements 3 and 4 are from two different genes targeted for suppression.  
     
     
         3 . A recombinant DNA construct of  claim 1  wherein the anti-sense-oriented DNA element 1 is from a 19-kD α-zein gene targeted for suppression, the anti-sense-oriented DNA element 2 is from a 22-kD α-zein gene targeted for suppression, the sense-oriented DNA element 3 is from a 22-kD α-zein gene targeted for suppression and is shorted than the anti-sense-oriented DNA element 2 and is complementary to only the 5′ end of element 2 and the sense-oriented DNA element 4 is from a 19-kD α-zein gene targeted for suppression and is complementary to at least a portion of the 5′ end of element 1.  
     
     
         4 . A recombinant DNA construct of  claim 1  wherein the more than one maize zein target gene is selected from the group consisting of 19- and 22-kD α-zeins, 16- and 27-kD γ-zeins, 10-kD δ-zeins, and 15-kD β-zeins.  
     
     
         5 . A recombinant DNA construct for suppression of more than one maize zein target gene that comprises in 5′ to 3′ order a promoter element operably linked to more than one anti-sense-oriented DNA element from more than one maize zein target gene, the anti-sense-oriented DNA elements designed in 5′ to 3′ order element 1, element 2, and at least one sense-oriented DNA element from at least one maize zein target gene, designated element 3, wherein the anti-sense-oriented DNA element 2 has at least a portion of its sequence at its 3′ end that is not complementary either to itself or to the sense-oriented DNA element 3, and sense-oriented RNA transcribed by the sense-oriented DNA element is complementary to the 5′-most end of anti-sense-oriented RNA transcribed by the anti-sense-oriented DNA element(s).  
     
     
         6 . A recombinant DNA construct of  claim 5  wherein the anti-sense-oriented DNA elements 1 and 2 are from two genes targeted for suppression and the sense-oriented DNA element 3 is from a gene targeted for suppression.  
     
     
         7 . A recombinant DNA construct of  claim 5  wherein the anti-sense-oriented DNA element 1 is from a 19-kD α-zein gene targeted for suppression, the anti-sense-oriented DNA element 2 is from a 22-kD α-zein gene targeted for suppression, and the sense-oriented DNA element 3 is from a 19-kD α-zein gene targeted for suppression.  
     
     
         8 . A recombinant DNA construct of  claim 5  wherein the more than one maize zein target gene is selected from the group consisting of 19- and 22-kD α-zeins, 16- and 27-kD γ-zeins, 10-kD δ-zeins, and 15-kD β-zeins and wherein the at least one maize zein target gene is selected from the group consisting of 19- and 22-kD α-zeins, 16- and 27-kD γ-zeins, 10-kD δ-zeins, and 15-kD βzeins.  
     
     
         9 . A method for generating corn seeds having at least one of enhanced lysine, enhanced oil and enhanced tryptophan comprising the steps of: 
 a) transforming a plant cell with the recombinant DNA construct of  claim 1  or  claim 4  for suppression of more than one maize zein target gene; and    b) regenerating the plant cell into a fertile transgenic plant, wherein the plant contains the construct in its genome; and    c) harvesting seed from the plant, wherein the seed has at least one of enhanced lysine, enhanced oil and enhanced tryptophan relative to seed of the same variety not transformed with the construct.    
     
     
         10 . A method for generating corn seeds having at least one of enhanced lysine, enhanced oil and enhanced tryptophan comprising the steps of: 
 d) transforming a plant cell with the recombinant DNA construct of  claim 5  or  claim 8  for suppression of more than one maize zein target gene; and    e) regenerating the plant cell into a fertile transgenic plant, wherein the plant contains the construct in its genome; and    f) harvesting seed from the plant, wherein the seed has at least one of enhanced lysine, enhanced oil and enhanced tryptophan relative to seed of the same variety not transformed with the construct.    
     
     
         11 . A transgenic plant comprising within its genome a DNA construct of  claim 1  or  claim 4  for suppression of more than one maize zein target gene.  
     
     
         12 . A harvested seed from a plant of  claim 11 .  
     
     
         13 . A harvested seed from a plant of  claim 11 , wherein the sense-oriented DNA elements are from two genes targeted for suppression and the anti-sense-oriented DNA elements are from two genes targeted for suppression.  
     
     
         14 . A processed product of the seed of  claim 12 , wherein the product is a feed, a meal, or a partially purified protein composition.  
     
     
         15 . A transgenic plant comprising within its genome a DNA construct of  claim 5  or  claim 8  for suppression of more than one maize zein target gene.  
     
     
         16 . A harvested seed from a plant of  claim 15 .  
     
     
         17 . A harvested seed from a plant of  claim 15 , wherein the sense-oriented DNA element is from one gene targeted for suppression and the anti-sense-oriented DNA elements are from two genes targeted for suppression.  
     
     
         18 . A processed product of the seed of  claim 16 , wherein the product is a feed, a meal, or a partially purified protein composition.