Synthetic brassica-derived chloroplast transit peptides

Abstract

This disclosure concerns compositions and methods for targeting peptides, polypeptides, and proteins to plastids of plastid-containing cells. In some embodiments, the disclosure concerns chloroplast transit peptides that may direct a polypeptide to a plastid, and nucleic acid molecules encoding the same. In some embodiments, the disclosure concerns methods for producing a transgenic plant material (e.g., a transgenic plant) comprising a chloroplast transit peptide, as well as plant materials produced by such methods, and plant commodity products produced therefrom.

What may be claimed is: 
     
         1 . An isolated nucleic acid molecule comprising a polynucleotide operably linked to a promoter that is functional in a plant cell, wherein the polynucleotide comprises:
 a nucleotide sequence encoding one synthetic chloroplast transit peptide (CTP), wherein the synthetic CTP consists of a contiguous amino acid sequence of a first CTP from a  Brassica  3-enolpyruvylshikimate-5-phosphate synthetase (EPSPS), and a contiguous amino acid sequence of a second, different CTP from a plant EPSPS; and   a nucleotide sequence encoding a plastid-targeted polypeptide,   wherein the polynucleotide encodes a chimeric polypeptide comprising the plastid-targeted polypeptide and the synthetic CTP.   
     
     
         2 . The isolated nucleic acid molecule of  claim 1 , wherein the contiguous amino acid sequence of the first CTP is positioned on the N-terminus of the synthetic CTP with respect to the position of the contiguous amino acid sequence of the second CTP. 
     
     
         3 . The isolated nucleic acid molecule of  claim 1 , wherein the first CTP is from a native polypeptide of  Brassica napus.    
     
     
         4 . The isolated nucleic acid molecule of  claim 1 , wherein the second CTP is from a polypeptide native to a  Brassica  species that is different from the  Brassica  species of the polypeptide of the first CTP. 
     
     
         5 . The isolated nucleic acid molecule of  claim 1 , wherein the second CTP is from a polypeptide native to an  Arabidopsis  plant. 
     
     
         6 . The isolated nucleic acid molecule of  claim 5 , wherein the second CTP is from a polypeptide native to  Arabidopsis thaliana.    
     
     
         7 . The isolated nucleic acid molecule of  claim 1 , wherein the synthetic CTP is not SEQ ID NO:1 or SEQ ID NO:3. 
     
     
         8 . The isolated nucleic acid molecule of  claim 1 , wherein the synthetic CTP is at least 80% identical to SEQ ID NO:6 or SEQ ID NO:8. 
     
     
         9 . The isolated nucleic acid molecule of  claim 1 , wherein the nucleotide sequence encoding the synthetic CTP is specifically hybridizable to an oligonucleotide consisting of a nucleotide sequence selected from the group consisting of SEQ ID NO:5 and SEQ ID NO:7. 
     
     
         10 . The isolated nucleic acid molecule of  claim 1 , wherein the polynucleotide is operably linked to at least one additional regulatory sequence. 
     
     
         11 . A chimeric polypeptide comprising:
 a synthetic chloroplast transit peptide (CTP) consisting of a contiguous amino acid sequence of a first CTP from a  Brassica  3-enolpyruvylshikimate-5-phosphate synthetase (EPSPS), and a contiguous amino acid sequence of a second, different CTP from a plant EPSPS; and   a plastid-targeted polypeptide.   
     
     
         12 . The chimeric polypeptide of  claim 11 , wherein the plastid-targeted polypeptide is targeted to a chloroplast when the chimeric polypeptide is expressed in a plant cell. 
     
     
         13 . The chimeric polypeptide of  claim 12 , wherein the synthetic CTP is removed when the plastid-targeted polypeptide is targeted to the chloroplast. 
     
     
         14 . The chimeric polypeptide of  claim 11 , wherein the plastid-targeted polypeptide is a fluorescent polypeptide. 
     
     
         15 . The chimeric polypeptide of  claim 11 , wherein the plastid-targeted polypeptide is an enzyme. 
     
     
         16 . The chimeric polypeptide of  claim 11 , wherein the plastid-targeted polypeptide is normally expressed in a plastid of a cell wherein the polypeptide is natively expressed. 
     
     
         17 . The chimeric polypeptide of  claim 11 , wherein the plastid-targeted polypeptide is selected from the group consisting of zeaxanthin epoxidase, choline monooxygenase, ferrochelatase, omega-3 fatty acid desaturase, glutamine synthetase, provitamin A, plant hormones, and Bt toxin proteins. 
     
     
         18 . The chimeric polypeptide of  claim 11 , wherein the plastid-targeted polypeptide increases the herbicide tolerance of a plant cell expressing the chimeric polypeptide. 
     
     
         19 . The chimeric polypeptide of  claim 18 , wherein the plastid-targeted polypeptide is selected from the group consisting of: acetolactase synthase (ALS), precursors of ALS, and EPSPS. 
     
     
         20 . The chimeric polypeptide of  claim 19 , wherein the plastid-targeted polypeptide does not contain a methionine at the first amino acid position. 
     
     
         21 . The nucleic acid molecule of  claim 1 , wherein the molecule is a plant expression vector. 
     
     
         22 . A transgenic plant material comprising the nucleic acid molecule of  claim 1 , wherein a cell of the plant material expresses the chimeric polypeptide from the polynucleotide. 
     
     
         23 . The transgenic plant material of  claim 22 , wherein the polynucleotide operably linked to the promoter is stably integrated into the genome of the cell of the plant material. 
     
     
         24 . The transgenic plant material of  claim 22 , wherein the plastid-targeted polypeptide is targeted to a chloroplast when the chimeric polypeptide is expressed in the cell of the plant material. 
     
     
         25 . The transgenic plant material of  claim 22 , wherein the plant material is selected from the group consisting of a plant tissue, a plant tissue culture, a callus culture, and a plant part. 
     
     
         26 . The transgenic plant material of  claim 22 , wherein the plant material is a plant cell. 
     
     
         27 . The transgenic plant material of  claim 22 , wherein the plant material is from a plant selected from the group consisting of  Arabidopsis,  alfalfa,  Brassica,  beans, broccoli, cabbage, carrot, cauliflower, celery, Chinese cabbage, cotton, cucumber, eggplant, lettuce, melon, pea, pepper, peanut, potato, pumpkin, radish, rapeseed, spinach, soybean, squash, sugarbeet, sunflower, tobacco, tomato, watermelon, corn, onion, rice, sorghum, wheat, rye, millet, sugarcane, oat, triticale, switchgrass, and turfgrass. 
     
     
         28 . A transgenic plant comprising the nucleic acid molecule of  claim 1 , wherein a cell of the plant expresses the chimeric polypeptide from the polynucleotide. 
     
     
         29 . The transgenic plant of  claim 28 , wherein the plant is selected from the group consisting of  Arabidopsis,  alfalfa,  Brassica,  beans, broccoli, cabbage, carrot, cauliflower, celery, Chinese cabbage, cotton, cucumber, eggplant, lettuce, melon, pea, pepper, peanut, potato, pumpkin, radish, rapeseed, spinach, soybean, squash, sugarbeet, sunflower, tobacco, tomato, watermelon, corn, onion, rice, sorghum, wheat, rye, millet, sugarcane, oat, triticale, switchgrass, and turfgrass. 
     
     
         30 . A method for producing a transgenic plant cell, the method comprising transforming the plant cell with the nucleic acid molecule of  claim 1 . 
     
     
         31 . The method according to  claim 30 , wherein the cell is transformed in a plant material selected from the group consisting of a plant tissue, a plant tissue culture, a callus culture, a plant part, and a whole plant. 
     
     
         32 . A method for producing a transgenic plant, the method comprising regenerating a transgenic plant from the transgenic plant material of  claim 25 . 
     
     
         33 . A transgenic plant commodity product produced from the transgenic plant material of  claim 25 , wherein the commodity product comprises a detectable amount of the polynucleotide, and wherein the commodity product is a food product, meal, oil, grain, or seed. 
     
     
         34 . An isolated nucleic acid molecule comprising a polynucleotide operably linked to a promoter that is functional in a plant cell, wherein the polynucleotide comprises:
 a  Brassica -derived means for targeting a polypeptide to a chloroplast; and   a nucleotide sequence encoding a plastid-targeted polypeptide, wherein the polynucleotide encodes a chimeric polypeptide comprising the plastid-targeted polypeptide and a peptide encoded by the  Brassica -derived means for targeting a polypeptide to a chloroplast.   
     
     
         35 . The nucleic acid molecule of  claim 34 , wherein the molecule is a plant transformation vector. 
     
     
         36 . A transgenic plant cell comprising the nucleic acid molecule of  claim 34 , wherein the plastid-targeted polypeptide is targeted to a chloroplast in the cell. 
     
     
         37 . The transgenic plant cell of  claim 36 , wherein the polynucleotide and promoter are stably integrated into the genome of the cell. 
     
     
         38 . A transgenic plant material comprising the transgenic plant cell of  claim 37 , wherein the plant material is selected from the group consisting of a plant tissue, a plant tissue culture, a callus culture, a plant part, and a whole plant. 
     
     
         39 . A method for producing a transgenic plant cell, the method comprising transforming a plant cell with the plant transformation vector of  claim 35 . 
     
     
         40 . The method according to  claim 39 , wherein the cell is transformed in a plant material selected from the group consisting of a plant tissue, a plant tissue culture, a callus culture, a plant part, and a whole plant.