Production of high tryptophan maize by chloroplast targeted expression of anthranilate synthase
View Patent ↗Novel expression vectors and constructs encoding a chloroplast transit peptide (CTP) operably linked to a monomeric anthranilate synthase are provided. Additionally, novel polynucleotide sequences encoding monomeric anthranilate synthases are provided. Also provided are methods for increasing the levels of free tryptophan in transgenic plants containing the expression vectors and constructs.
1. An expression vector comprising a polynucleotide molecule encoding a chloroplast transit peptide fused to a monomeric anthranilate synthase, wherein the chloroplast transit peptide is heterologous to the monomeric anthranilate synthase, wherein:
a) the chloroplast transit peptide comprises the polypeptide sequence of SEQ ID NO: 28; or
b) the polynucleotide molecule encoding the chloroplast transit peptide comprises SEQ ID NO: 9;
wherein said chloroplast transit peptide provides enhanced targeting of said monomeric anthranilate synthase to a chloroplast of a plant cell when compared to the targeting of said anthranilate synthase by chloroplast transit peptide 1 (CTP1) of SEQ ID NO:19;
wherein said polynucleotide molecule provides enhanced levels of at least tryptophan in a transgenic plant cell comprising said polynucleotide molecule when compared to a plant cell of the same genotype but lacking said polynucleotide molecule; and wherein said monomeric anthranilate synthase is feedback insensitive to tryptophan.
2. The expression vector of claim 1 , wherein said monomeric anthranilate synthase comprises a polypeptide sequence comprising SEQ ID NO: 206.
3. The expression vector of claim 1 , defined as comprising the nucleic acid sequence of SEQ ID NO: 213.
4. The expression vector of claim 1 , further comprising a promoter functional in plants operably linked to said polynucleotide encoding the chloroplast transit peptide fused to the monomeric anthranilate synthase.
5. The expression vector of claim 4 , wherein said promoter is a constitutive, inducible, seed-specific, or tissue-preferred promoter.
6. The expression vector of claim 4 wherein the polynucleotide encoding the chloroplast transit peptide comprises SEQ ID NO: 9, and the polynucleotide encoding the monomeric anthranilate synthase comprises SEQ ID NO: 207.
7. A transgenic cell transformed with the expression vector of claim 1 .
8. The transgenic cell of claim 7 , defined as a plant cell.
9. The transgenic plant cell of claim 8 , defined as a monocot cell.
10. The transgenic plant cell of claim 8 , defined as a dicot cell.
11. The transgenic plant cell of claim 8 , wherein said monomeric anthranilate synthase comprises a polypeptide sequence comprising SEQ ID NO: 206.
12. The transgenic plant cell of claim 8 , further comprising a promoter operably linked to said polynucleotide molecule encoding said chloroplast transit peptide fused to said monomeric anthranilate synthase.
13. The transgenic plant cell of claim 12 , wherein said promoter is selected from the group consisting of constitutive, inducible, seed-specific, and tissue-preferred promoters.
14. The transgenic plant cell of claim 8 , wherein said transgenic plant cell is a maize cell.
15. The transgenic plant cell of claim 8 , wherein the polynucleotide encoding the chloroplast transit peptide comprises SEQ ID NO: 9 and the polynucleotide encoding the monomeric anthranilate synthase comprises SEQ ID NO: 207.
16. A method of increasing the levels of free tryptophan in a cell of a monocotyledonous plant comprising expressing in the cell the expression vector of claim 1 , wherein said chloroplast transit peptide compartmentalizes said monomeric anthranilate synthase to a plastid in said cell.
17. A transgenic plant transformed with the expression vector of claim 1 .
18. A seed of the plant of claim 17 , wherein the seed comprises said expression vector.
19. A method of producing a tryptophan-enhanced corn feed product comprising processing the seed of claim 18 into said corn feed product.