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 is claimed is: 
     
         1 . A nucleic acid molecule comprising a polynucleotide that encodes a chimeric protein comprising a synthetic chloroplast transit peptide (CTP) and a linked 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) polypeptide,
 wherein the amino acid sequence of the synthetic CTP selected from the group consisting of:
 (A) a first amino acid sequence between 25 and 41 amino acids in length, wherein the first amino acid sequence is at least 90% identical to a contiguous amino acid sequence of the same length in the first 41 amino acids of SEQ ID NO:1, linked to a second amino acid sequence between 25 and 41 amino acids in length, wherein the second amino acid sequence is at least 90% identical to a contiguous amino acid sequence of the same length in the last 41 amino acids of SEQ ID NO:2, and 
 (B) a first amino acid sequence between 25 and 41 amino acids in length, wherein the first amino acid sequence is at least 90% identical to a contiguous amino acid sequence of the same length in the first 41 amino acids of SEQ ID NO:2, linked to a second amino acid sequence between 25 and 41 amino acids in length, wherein the second amino acid sequence is at least 90% identical to a contiguous amino acid sequence of the same length in the last 41 amino acids of SEQ ID NO:1; and 
   wherein the EPSPS polypeptide comprises the amino acid sequences of [SEQ ID NOs:170-173 from P11743.1US].   
     
     
         2 . The nucleic acid molecule of  claim 1 , wherein the EPSPS polypeptide comprises an amino acid sequence that is at least 90% identical to [DGT-28, DGT-31, DGT-32, or DGT-33]. 
     
     
         3 . The nucleic acid molecule of  claim 2 , wherein the EPSPS polypeptide comprises an amino acid sequence that is at least 95% identical to [DGT-28, DGT-31, DGT-32, or DGT-33]. 
     
     
         4 . The nucleic acid molecule of  claim 2 , wherein the chimeric protein comprises the amino acid sequence encoded by SEQ ID NO:34 or SEQ ID NO:35. 
     
     
         5 . The nucleic acid molecule of  claim 1 , wherein the synthetic CTP comprises SEQ ID NO:13. 
     
     
         6 . The nucleic acid molecule of  claim 1 , wherein the synthetic CTP is less than 70 amino acids in length. 
     
     
         7 . The nucleic acid molecule of  claim 1 , wherein the synthetic CTP is about 65 amino acids in length. 
     
     
         8 . The nucleic acid molecule of  claim 2 , wherein the synthetic CTP comprises less than 7 conservative amino acid substitutions to the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4, such that the synthetic CTP is at least 90% identical to SEQ ID NO:3 or SEQ ID NO:4. 
     
     
         9 . The nucleic acid molecule of  claim 2 , wherein the synthetic CTP comprises less than 4 conservative amino acid substitutions to the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4, such that the synthetic CTP is at least 95% identical to SEQ ID NO:3 or SEQ ID NO:4. 
     
     
         10 . The nucleic acid molecule of  claim 2 , wherein the synthetic CTP comprises 1 conservative amino acid substitution to the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4, such that the synthetic CTP is at least 98% identical to SEQ ID NO:3 or SEQ ID NO:4. 
     
     
         11 . The nucleic acid molecule of  claim 2 , wherein the first amino acid sequence of the CTP comprises amino acids 1-35 of SEQ ID NO:1 or amino acids 1-31 of SEQ ID NO:2. 
     
     
         12 . The nucleic acid molecule of  claim 2 , wherein the second amino acid sequence of the CTP comprises amino acids 32-61 of SEQ ID NO:2 or amino acids 36-69 of SEQ ID NO:1. 
     
     
         13 . The nucleic acid molecule of  claim 2 , wherein the polynucleotide is operably linked to a promoter operable in a plant cell. 
     
     
         14 . The nucleic acid molecule of  claim 8 , wherein the polynucleotide is operably linked to a promoter operable in a plant cell. 
     
     
         15 . The nucleic acid molecule of  claim 14 , wherein the molecule is a plant expression vector. 
     
     
         16 . A transgenic plant material comprising the nucleic acid molecule of  claim 14  as a genomic nucleic acid molecule, wherein the EPSPS polypeptide is targeted to chloroplasts of the plant material. 
     
     
         17 . The transgenic plant material of  claim 16 , wherein the plant material is selected from the group consisting of a plant cell, a plant tissue, a plant tissue culture, a callus culture, and a plant part. 
     
     
         18 . The transgenic plant material of  claim 16 , wherein the plant material is a whole plant or seed. 
     
     
         19 . The transgenic plant material of  claim 18 , 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. 
     
     
         20 . A method for producing a transgenic plant material, the method comprising transforming a plant material with the nucleic acid molecule of  claim 15 . 
     
     
         21 . The method according to  claim 20 , wherein the plant material is selected from the group consisting of a plant cell, a plant tissue, a plant tissue culture, and a callus culture. 
     
     
         22 . A transgenic plant commodity product produced from the plant material of  claim 16 , wherein the commodity product comprises the polynucleotide. 
     
     
         23 . A method for growing a glyphosate-tolerant plant, the method comprising:
 planting or cultivating the transgenic plant or seed of  claim 18  in a suitable growing environment.   
     
     
         24 . The method according to  claim 23 , the method further comprising:
 applying glyphosate to the growing environment.