Increased protein expression in plants
This disclosure concerns synthetic polynucleotides encoding a polypeptide of interest that are particularly well-suited for expression in target plants.
1. A system for expressing a heterologous polypeptide of interest in a cell of a maize plant, the system comprising:
a reference nucleotide sequence encoding the polypeptide of interest from the genome of an organism other than the maize plant; and
a synthetic polynucleotide comprising:
a nucleotide sequence encoding the polypeptide of interest, wherein the nucleotide sequence has been codon-optimized to remove codons selected from the group consisting of TTA, CTA, GTA, CGT, AGT, and CGA from the reference nucleotide,
wherein the synthetic polynucleotide nucleotide sequence encoding the polypeptide of interest comprises at least one polyadenylation sequence selected from the group consisting of AATAAA, AATAAT, AACCAA, ATATAA, AATCAA, ATACTA, ATAAAA, ATGAAA, AAGCAT, ATTAAT, ATACAT, AAAATA, ATTAAA, AATTAA, AATACA, and CATAAA, present in the same number and in the same location as in the reference nucleotide sequence,
wherein the synthetic polynucleotide nucleotide sequence encoding the polypeptide of interest does not comprise any polyadenylation sequence selected from the group consisting of AATAAT, AACCAA, ATATAA, ATACTA, ATAAAA, ATGAAA, AAGCAT, ATTAAT, ATACAT, AAAATA, ATTAAA, AATTAA, AATACA, and CATAAA in a different number or location than in the reference nucleotide sequence, and
wherein the synthetic polynucleotide nucleotide sequence encoding the polypeptide of interest does not comprise a polyadenylation sequence selected from the group consisting of ATATAT, TTGTTT, TTTTGT, TGTTTT, TATATA, TATTTT, TTTTTT, ATTTTT, TTATTT, TTTATT, TAATAA, ATTTAT, TATATT, TTTTAT, ATATTT, TATTAT, TGTTTG, TTATAT, TGTAAT, AAATAA, AATTTT, TTTTTA, TAATTT, TTAATT, AAATTT, TTTGTT, ATTATT, ATTTTA, TTTAAT, and TTTTAA.
2. The system of claim 1 , wherein the nucleotide sequence encoding the polypeptide of interest has been codon-optimized to remove from the reference nucleotide sequence all codons selected from the group consisting of TTA, CTA, GTA, CGT, AGT, and CGA.
3. The system of claim 1 , wherein the polypeptide of interest is a protein selected from the group consisting of insecticidal proteins, herbicide tolerance proteins, stress tolerance-related proteins, and oil profile modification proteins.
4. The system of claim 1 , wherein the polypeptide of interest is an insecticidal protein.
5. The system of claim 4 , wherein the polypeptide of interest is Cry 1 Ab or Cry1Fa.
6. The system of claim 5 , wherein the reference nucleotide sequence is SEQ ID NO:30.
7. The system of claim 5 , wherein the reference nucleotide sequence is SEQ ID NO:31.
8. A transgenic maize plant comprising a synthetic polynucleotide comprising:
a nucleotide sequence encoding a polypeptide of interest, wherein the nucleotide sequence has been codon-optimized from a reference nucleotide sequence encoding the polypeptide of interest from the genome of an organism other than maize to remove codons selected from the group consisting of TTA, CTA, GTA, CGT, AGT, and CGA,
wherein the synthetic polynucleotide nucleotide sequence encoding the polypeptide of interest comprises at least one polyadenylation sequence selected from the group consisting of AATAAA, AATAAT, AACCAA, ATATAA, AATCAA, ATACTA, ATAAAA, ATGAAA, AAGCAT, ATTAAT, ATACAT, AAAATA, ATTAAA, AATTAA, AATACA, and CATAAA, present in the same number and in the same location as in the reference nucleotide sequence,
wherein the synthetic polynucleotide nucleotide sequence encoding the polypeptide of interest does not comprise any polyadenylation sequence selected from the group consisting of AATAAT, AACCAA, ATATAA, ATACTA, ATAAAA, ATGAAA, AAGCAT, ATTAAT, ATACAT, AAAATA, ATTAAA, AATTAA, AATACA, and CATAAA in a different number or location than in the reference nucleotide sequence, and
wherein the synthetic polynucleotide nucleotide sequence encoding a polypeptide of interest does not comprise a polyadenylation sequence selected from the group consisting of ATATAT, TTGTTT, TTTTGT, TGTTTT, TATATA, TATTTT, TTTTTT, ATTTTT, TTATTT, TTTATT, TAATAA, ATTTAT, TATATT, TTTTAT, ATATTT, TATTAT, TGTTTG, TTATAT, TGTAAT, AAATAA, AATTTT, TTTTTA, TAATTT, TTAATT, AAATTT, TTTGTT, ATTATT, ATTTTA, TTTAAT, and TTTTAA.
9. The transgenic maize plant of claim 8 , wherein the nucleotide sequence encoding the polypeptide of interest has been codon-optimized from the reference nucleotide sequence to remove all codons selected from the group consisting of TTA, CTA, GTA, CGT, AGT, and CGA.
10. The transgenic maize plant of claim 8 , wherein the polypeptide of interest is a protein selected from the group consisting of insecticidal proteins, herbicide tolerance proteins, stress tolerance-related proteins, and oil profile modification proteins.
11. The transgenic maize plant of claim 8 , wherein the polypeptide of interest is an insecticidal protein.
12. The transgenic maize plant of claim 11 , wherein the polypeptide of interest is Cry1Ab or Cry1Fa.
13. The transgenic maize plant of claim 12 , wherein the reference nucleotide sequence is SEQ ID NO:30.
14. The transgenic maize plant of claim 12 , wherein the reference nucleotide sequence is SEQ ID NO:31.
15. A composition derived from the transgenic maize plant of claim 8 , wherein the composition is a commodity product selected from the group consisting of meal, flour, protein concentrate, and oil.
16. A transgenic maize plant cell comprising a synthetic polynucleotide comprising:
a nucleotide sequence encoding a polypeptide of interest, wherein the nucleotide sequence has been codon-optimized from a reference nucleotide sequence encoding the polypeptide of interest from the genome of an organism other than maize to remove codons selected from the group consisting of TTA, CTA, GTA, CGT, AGT, and CGA,
wherein the synthetic polynucleotide nucleotide sequence encoding the polypeptide of interest comprises at least one polyadenylation sequence selected from the group consisting of AATAAA, AATAAT, AACCAA, ATATAA, AATCAA, ATACTA, ATAAAA, ATGAAA, AAGCAT, ATTAAT, ATACAT, AAAATA, ATTAAA, AATTAA, AATACA, and CATAAA, present in the same number and in the same location as in the reference nucleotide sequence,
wherein the synthetic polynucleotide nucleotide sequence encoding the polypeptide of interest does not comprise any polyadenylation sequence selected from the group consisting of AATAAT, AACCAA, ATATAA, ATACTA, ATAAAA, ATGAAA, AAGCAT, ATTAAT, ATACAT, AAAATA, ATTAAA, AATTAA, AATACA, and CATAAA in a different number or location than in the reference nucleotide sequence, and
wherein the synthetic polynucleotide nucleotide sequence encoding the polypeptide of interest does not comprise a polyadenylation sequence selected from the group consisting of ATATAT, TTGTTT, TTTTGT, TGTTTT, TATATA, TATTTT, TTTTTT, ATTTTT, TTATTT, TTTATT, TAATAA, ATTTAT, TATATT, TTTTAT, ATATTT, TATTAT, TGTTTG, TTATAT, TGTAAT, AAATAA, AATTTT, TTTTTA, TAATTT, TTAATT, AAATTT, TTTGTT, ATTATT, ATTTTA, TTTAAT, and TTTTAA.
17. The transgenic maize plant cell of claim 16 , wherein the nucleotide sequence encoding the polypeptide of interest has been codon-optimized from the reference nucleotide sequence to remove all codons selected from the group consisting of TTA, CTA, GTA, CGT, AGT, and CGA.
18. The transgenic maize plant cell of claim 16 , wherein the polypeptide of interest is a protein selected from the group consisting of insecticidal proteins, herbicide tolerance proteins, stress tolerance-related proteins, and oil profile modification proteins.
19. The transgenic maize plant cell of claim 16 , wherein the polypeptide of interest is an insecticidal protein.
20. The transgenic maize plant cell of claim 19 , wherein the polypeptide of interest is Cry1Ab or Cry1Fa.
21. The transgenic maize plant cell of claim 20 , wherein the reference nucleotide sequence is SEQ ID NO:30.
22. The transgenic maize plant cell of claim 20 , wherein the reference nucleotide sequence is SEQ ID NO:31.
23. A plant material comprising the transgenic maize plant cell of claim 16 , wherein the plant material comprises a detectable level of the synthetic polynucleotide.
24. A system for expressing a heterologous polypeptide of interest in a cell of a soybean plant, the system comprising:
a reference nucleotide sequence encoding the polypeptide of interest from the genome of an organism other than the soybean plant; and
a synthetic polynucleotide comprising:
a nucleotide sequence encoding the polypeptide of interest, wherein the sequence has been codon-optimized to remove from the reference nucleotide sequence all codons selected from the group consisting of TTA, CTA, TCG, CCG, ACG, GCG, CGA, and CGG,
wherein the synthetic polynucleotide nucleotide sequence encoding the polypeptide of interest comprises at least one polyadenylation sequence selected from the group consisting of AATAAA, AATAAT, AACCAA, ATATAA, AATCAA, ATACTA, ATAAAA, ATGAAA, AAGCAT, ATTAAT, ATACAT, AAAATA, ATTAAA, AATTAA, AATACA, and CATAAA, present in the same number and in the same location as in the reference nucleotide sequence,
wherein the synthetic polynucleotide nucleotide sequence encoding the polypeptide of interest does not comprise any polyadenylation sequence selected from the group consisting of AATAAT, AACCAA, ATATAA, ATACTA, ATAAAA, ATGAAA, AAGCAT, ATTAAT, ATACAT, AAAATA, ATTAAA, AATTAA, AATACA, and CATAAA in a different number or location than in the reference nucleotide sequence, and
wherein the synthetic polynucleotide nucleotide sequence encoding the polypeptide of interest does not comprise a polyadenylation sequence selected from the group consisting of ATATAT, TTGTTT, TTTTGT, TGTTTT, TATATA, TATTTT, TTTTTT, ATTTTT, TTATTT, TTTATT, TAATAA, ATTTAT, TATATT, TTTTAT, ATATTT, TATTAT, TGTTTG, TTATAT, TGTAAT, AAATAA, AATTTT, TTTTTA, TAATTT, TTAATT, AAATTT, TTTGTT, ATTATT, ATTTTA, TTTAAT, and TTTTAA.
25. The system of claim 24 , wherein the polypeptide of interest is a protein selected from the group consisting of insecticidal proteins, herbicide tolerance proteins, stress tolerance-related proteins, and oil profile modification proteins.
26. The system of claim 24 , wherein the polypeptide of interest is an insecticidal protein.
27. The system of claim 26 , wherein the polypeptide of interest is Cry1Ab or Cry1Fa.
28. The system of claim 27 , wherein the reference nucleotide sequence is SEQ ID NO:30.
29. The system of claim 27 , wherein the reference nucleotide sequence is SEQ ID NO:31.
30. A transgenic soybean plant comprising a synthetic polynucleotide comprising:
a nucleotide sequence encoding a polypeptide of interest, wherein the nucleotide sequence has been codon-optimized from a reference nucleotide sequence encoding the polypeptide of interest from the genome of an organism other than soybean to remove all codons selected from the group consisting of TTA, CTA, TCG, CCG, ACG, GCG, CGA, and CGG,
wherein the synthetic polynucleotide nucleotide sequence encoding the polypeptide of interest comprises at least one polyadenylation sequence selected from the group consisting of AATAAA, AATAAT, AACCAA, ATATAA, AATCAA, ATACTA, ATAAAA, ATGAAA, AAGCAT, ATTAAT, ATACAT, AAAATA, ATTAAA, AATTAA, AATACA, and CATAAA, present in the same number and in the same location as in the reference nucleotide sequence,
wherein the synthetic polynucleotide nucleotide sequence encoding the polypeptide of interest does not comprise any polyadenylation sequence selected from the group consisting of AATAAT, AACCAA, ATATAA, ATACTA, ATAAAA, ATGAAA, AAGCAT, ATTAAT, ATACAT, AAAATA, ATTAAA, AATTAA, AATACA, and CATAAA in a different number or location than in the reference polynucleotide, and
wherein the sequence of the synthetic polynucleotide does not comprise a polyadenylation sequence selected from the group consisting of ATATAT, TTGTTT, TTTTGT, TGTTTT, TATATA, TATTTT, TTTTTT, ATTTTT, TTATTT, TTTATT, TAATAA, ATTTAT, TATATT, TTTTAT, ATATTT, TATTAT, TGTTTG, TTATAT, TGTAAT, AAATAA, AATTTT, TTTTTA, TAATTT, TTAATT, AAATTT, TTTGTT, ATTATT, ATTTTA, TTTAAT, and TTTTAA.
31. The transgenic soybean plant of claim 30 , wherein the polypeptide of interest is a protein selected from the group consisting of insecticidal proteins, herbicide tolerance proteins, stress tolerance-related proteins, and oil profile modification proteins.
32. The transgenic soybean plant of claim 30 , wherein the polypeptide of interest is an insecticidal protein.
33. The transgenic soybean plant of claim 32 , wherein the polypeptide of interest is Cry1Ab or Cry1Fa.
34. The transgenic soybean plant of claim 33 , wherein the reference nucleotide sequence is SEQ ID NO:30.
35. The transgenic soybean plant of claim 33 , wherein the reference nucleotide sequence is SEQ ID NO:31.
36. A composition derived from the transgenic soybean plant of claim 30 , wherein the composition is a commodity product selected from the group consisting of meal, flour, protein concentrate, and oil.
37. A transgenic soybean plant cell comprising a synthetic polynucleotide comprising:
a nucleotide sequence encoding a polypeptide of interest, wherein the nucleotide sequence has been codon-optimized from a reference nucleotide sequence encoding the polypeptide of interest from the genome of an organism other than soybean to remove all codons selected from the group consisting of TTA, CTA, TCG, CCG, ACG, GCG, CGA, and CGG,
wherein the synthetic polynucleotide nucleotide sequence encoding the polypeptide of interest comprises at least one polyadenylation sequence selected from the group consisting of AATAAA, AATAAT, AACCAA, ATATAA, AATCAA, ATACTA, ATAAAA, ATGAAA, AAGCAT, ATTAAT, ATACAT, AAAATA, ATTAAA, AATTAA, AATACA, and CATAAA, present in the same number and in the same location as in the reference nucleotide sequence,
wherein the synthetic polynucleotide nucleotide sequence encoding the polypeptide of interest does not comprise any polyadenylation sequence selected from the group consisting of AATAAT, AACCAA, ATATAA, ATACTA, ATAAAA, ATGAAA, AAGCAT, ATTAAT, ATACAT, AAAATA, ATTAAA, AATTAA, AATACA, and CATAAA in a different number or location than in the reference nucleotide sequence, and
wherein the synthetic polynucleotide nucleotide sequence encoding the polypeptide of interest does not comprise a polyadenylation sequence selected from the group consisting of ATATAT, TTGTTT, TTTTGT, TGTTTT, TATATA, TATTTT, TTTTTT, ATTTTT, TTATTT, TTTATT, TAATAA, ATTTAT, TATATT, TTTTAT, ATATTT, TATTAT, TGTTTG, TTATAT, TGTAAT, AAATAA, AATTTT, TTTTTA, TAATTT, TTAATT, AAATTT, TTTGTT, ATTATT, ATTTTA, TTTAAT, and TTTTAA.
38. The transgenic soybean plant cell of claim 37 , wherein the polypeptide of interest is a protein selected from the group consisting of insecticidal proteins, herbicide tolerance proteins, stress tolerance-related proteins, and oil profile modification proteins.
39. The transgenic soybean plant cell of claim 37 , wherein the polypeptide of interest is an insecticidal protein.
40. The transgenic soybean plant cell of claim 39 , wherein the polypeptide of interest is Cry1Ab or Cry1Fa.
41. The transgenic soybean plant cell of claim 40 , wherein the reference nucleotide sequence is SEQ ID NO:30.
42. The transgenic maize plant cell of claim 40 , wherein the reference nucleotide sequence is SEQ ID NO:31.
43. A plant material comprising the transgenic soybean plant cell of claim 37 , wherein the plant material comprises a detectable level of the synthetic polynucleotide.