Methods and Compositions for Selective Regulation of Protein Expression

Abstract

The invention provides methods and compositions for selectively suppressing the expression of a recombinant protein in a male reproductive tissue of a transgenic plant. The invention also provides methods and compositions for inducing male sterility in a transgenic plant. Plants, plant cells, plant parts, seeds, and commodity products including such compositions are aspects of the invention.

1 . A recombinant DNA construct comprising a protein-coding sequence operably linked to a DNA sequence comprising a male tissue-specific siRNA (mts-siRNA) element, wherein said mts-siRNA element is heterologous with respect to said protein-coding sequence, and wherein said protein-coding sequence encodes a herbicide tolerance protein or a pest resistance protein. 
     
     
         2 . The recombinant DNA construct of  claim 1 , wherein said mts-siRNA element comprises at least one mts-siRNA sequence. 
     
     
         3 . The recombinant DNA construct of  claim 1 , wherein said mts-siRNA element comprises a fragment of a nucleotide sequence selected from the group consisting of SEQ ID NO:1-[[56]]94 and [[105]]96-149, wherein said fragment comprises at least 18 contiguous nucleotides. 
     
     
         4 . The recombinant DNA construct of  claim 1 , wherein said protein-coding sequence is a pest resistance protein, and wherein said mts-siRNA element is selected from the group consisting of SEQ ID NO:88 and 89. 
     
     
         5 . The recombinant DNA construct of  claim 1 , wherein the expression of said protein-coding sequence in a transgenic plant confers at least vegetative herbicide tolerance to said plant. 
     
     
         6 . The recombinant DNA construct of  claim 5 , wherein said protein-coding sequence encodes a glyphosate-tolerant EPSPS. 
     
     
         7 . A method of making a recombinant DNA construct comprising operably linking a polynucleotide sequence comprising a mts-siRNA element to a protein-coding sequence, wherein said mts-siRNA element is heterologous with respect to said protein-coding sequence, and wherein said protein-coding sequence encodes a herbicide tolerance or a pest resistance protein. 
     
     
         8 . The method of  claim 7 , wherein said at least one mts-siRNA element comprises a fragment of a nucleotide sequence is at least one selected from the group consisting of SEQ ID NO:1-[[56]]94 and [[105]]96-149, and wherein said fragment comprises at least 18 contiguous nucleotides. 
     
     
         9 . The method of  claim 7 , wherein said mts-siRNA element is tassel-specific. 
     
     
         10 . A transgenic plant having in its genome the recombinant DNA construct of  claim 1 . 
     
     
         11 . A seed, progeny, or plant part of the transgenic plant of  claim 10 . 
     
     
         12 . The transgenic plant of  claim 10 , wherein said transgenic plant is a monocotyledonous plant. 
     
     
         13 . The transgenic plant of  claim 10 , wherein said transgenic plant is a maize plant. 
     
     
         14 . A method of selectively suppressing the expression of a recombinant protein in a male reproductive tissue of a transgenic plant comprising expressing in said transgenic plant a recombinant DNA construct of  claim 1 . 
     
     
         15 . The method of  claim 14 , wherein said male reproductive tissue is a tassel of a maize plant. 
     
     
         16 . The method of  claim 14 , wherein said mts-siRNA element comprises at least three mts-siRNA sequences. 
     
     
         17 . The method of  claim 14 , wherein said mts-siRNA element comprises a fragment of a nucleotide sequence selected from the group consisting of SEQ ID NO:1[[56]]94 and [[105]]96-149, wherein said fragment comprises at least 18 contiguous nucleotides. 
     
     
         18 . The method of  claim 14 , wherein said wherein said protein-coding sequence is a pest resistance protein, and wherein said mts-siRNA element is selected from the group consisting of SEQ ID NO:88 and 89. 
     
     
         19 . The method of  claim 14 , wherein the expression of said recombinant protein in a transgenic plant confers at least vegetative herbicide tolerance to said plant. 
     
     
         20 . The method of  claim 19 , wherein said recombinant protein is a glyphosate-tolerant EPSPS. 
     
     
         21 . A method of inducing male-sterility in a transgenic plant comprising applying an effective amount of an herbicide to a transgenic plant comprising a recombinant DNA construct of  claim 1 , wherein said herbicide application is carried out during the development of the male reproductive tissue of said transgenic plant thereby inducing male-sterility in said transgenic plant. 
     
     
         22 . The method of  claim 21 , wherein said transgenic plant is a maize plant. 
     
     
         23 . The method of  claim 21 , wherein said herbicide application prevents at least pollen shed or anther extrusion. 
     
     
         24 . The method of  claim 21 , wherein said development of the male reproductive tissue is a stage selected from the group consisting of the V4, V5, V6, V7, V8, V9, V10, V11, V12, V13, and V14 stage of maize plant development. 
     
     
         25 . The method of  claim 21 , wherein said herbicide is selected from the group consisting of acetyl coenzyme A carboxylase (ACCase) inhibitors, acetolactate synthase (ALS) inhibitors, photosystem II (PSII) inhibitors, protoporphyrinogen oxidase (PPO) inhibitors, 4-hydroxyphenyl pyruvate dioxygenase (HPPD) inhibitors, 5enolypyruvyl shikimate 3-phosphate synthase (EPSPS) inhibitors, glutamine synthetase (GS) inhibitors, and synthetic auxins. 
     
     
         26 . The method of  claim 21 , wherein said herbicide is glyphosate and said recombinant protein is a glyphosate-tolerant EPSPS. 
     
     
         27 . A method of producing hybrid seed comprising:
 a. applying an effective amount of herbicide to a transgenic plant comprising a recombinant DNA construct of  claim 1 , wherein said herbicide application is carried out during the development of the male reproductive tissue of said transgenic plant thereby inducing male-sterility in said transgenic plant;   b. fertilizing said transgenic plant with pollen from a second plant; and   c. harvesting hybrid seed from said transgenic plant.   
     
     
         28 . The method of  claim 27 , wherein said transgenic plant is maize. 
     
     
         29 . The method of  claim 27 , wherein said herbicide is glyphosate and said recombinant protein is a glyphosate-tolerant EPSPS. 
     
     
         30 . The method of  claim 29 , wherein said glyphosate is applied during said development at an effective dose of about 0.125 pounds acid equivalent per acre to about 8 pounds acid equivalent per acre. 
     
     
         31 . Hybrid seed harvested from a male-sterile transgenic plant that has been fertilized with pollen from a second plant, wherein said male-sterile transgenic plant comprises a recombinant DNA construct of  claim 1  and has been induced to be male-sterile by application of an effective amount of herbicide during the development of the male reproductive tissue of said transgenic plant.