Regulatory sequences for expression of transgenes
The invention provides important tools for the use of Chlorophyte organisms, including regulatory sequences useful for the expression of transgenes in such organisms. Also provided are vectors and expression cassettes containing promoters and/or terminators disclosed herein for expression of transgenes in Chlorophyte organisms. Methods of using these tools are also provided.
1. A nucleotide sequence comprising a promoter having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of: SEQ ID NO: 1, 3, 5, 7, 9, and 11, and operably linked to a heterologous nucleotide sequence of interest.
2. A vector for transforming a microorganism comprising the nucleotide sequence of claim 1 .
3. A microorganism comprising the nucleotide sequence of claim 1 .
4. The microorganism of claim 3 , wherein said microorganism is an algal microorganism that expresses the heterologous nucleotide sequence of interest.
5. The microorganism of claim 4 , wherein said microorganism is a Chlorophyte microorganism.
6. The microorganism of claim 5 , wherein the Chlorophyte microorganism is of the Class Trebouxiophyceae.
7. The microorganism of claim 6 , wherein the Trebouxiophyceae microorganism is Picochlorum sp.
8. The nucleotide sequence of claim 1 , further comprising a terminator sequence.
9. An expression cassette comprising the nucleotide sequence of claim 8 .
10. The nucleotide sequence of claim 8 , wherein said terminator sequence has at least 90% sequence identity to a terminator sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, and 12.
11. The nucleotide sequence of claim 8 wherein:
a. the promoter sequence has at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 1 and the terminator sequence has at least 90 % sequence identity to the nucleotide sequence of SEQ ID NO: 2; or
b. the promoter sequence has at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 3 and the terminator sequence hast at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 4; or
c. the promoter sequence has at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 5 and the terminator sequence hast at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 6; or
d. the promoter sequence has at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 7 and the terminator sequence hast at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 8.
12. The nucleotide sequence of claim 8 wherein:
a. the promoter sequence has at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 9 and the terminator sequence has at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 10; or
b. the promoter sequence has at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 11 and the terminator sequence hast at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 12.
13. A vector for transforming a microorganism comprising the nucleotide sequence of claim 11 .
14. A microorganism comprising the vector of claim 13 , wherein said microorganism is an alga.
15. The microorganism of claim 14 , wherein said microorganism is a Trebouxiophyte microorganism.
16. An expression cassette comprising the nucleotide sequence of claim 10 .
17. A method of transforming an algal cell comprising introducing a vector comprising the expression cassette of claim 16 into an algal cell and selecting for a transformant comprising said vector.
18. The nucleotide sequence of claim 1 , wherein the heterologous sequence of interest encodes a polypeptide or a functional RNA.
19. A nucleotide sequence comprising a promoter having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of: SEQ ID NO: 1, 3, 5, 7, 9, and 11, and operably linked to a nucleotide sequence encoding a protein or a functional RNA heterologous to Picochlorum sp.
20. The nucleotide sequence of claim 19 , further comprising a nucleotide sequence having at least 90% sequence identity to a terminator sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, and 12.
21. An expression cassette comprising the nucleotide sequence of claim 20 .
22. An expression cassette comprising the nucleotide sequence of claim 20 , wherein:
a. the promoter sequence has at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 1 and the terminator sequence has at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 2; or
b. the promoter sequence has at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 3 and the terminator sequence hast at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 4; or
c. the promoter sequence has at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 5 and the terminator sequence hast at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 6; or
d. the promoter sequence has at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 7 and the terminator sequence hast at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 8.
23. The nucleotide sequence of claim 11 , wherein the promoter sequence has at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 1 and the terminator sequence has at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 2.
24. The nucleotide sequence of claim 11 , wherein the promoter sequence has at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 7 and the terminator sequence has at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 8.