Nannochloropsis spliced leader sequences and uses therefor
The present invention relates to the culture and manipulation of microorganisms for biotech applications, and is based on the discovery and characterization of spliced leader sequences identified in transcripts from Nannochloropsis species. In particular, the invention provides nucleic acid compositions comprising a SL sequence operably linked to a protein-encoding gene. Further provided are compositions and methods for enhanced gene expression in recombinant microorganisms as well as methods for identification and/or isolation of nucleic acid molecules tagged with a spliced leader sequence.
1. A method of producing cDNA, comprising:
hybridizing at least one 3′ primer to a population of RNA molecules isolated from a cell, wherein the at least one 3′ primer is selected from the group consisting of: a population of random primers, a degenerate primer, one or more gene-specific primers, one or more gene family-specific primers, and a primer comprising oligo-dT, to generate a population of RNA molecules hybridized to the at least one 3′ primer;
synthesizing a population of first strands of cDNA from the population of RNA molecules hybridized to the at least one 3′ primer using at least one polymerase;
hybridizing a 5′ primer comprising a nucleic acid sequence having at least 83% identity to a SL sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4 to the population of first strands of cDNA; and
synthesizing second strands of cDNA from the population of first strands of cDNA hybridized to the 5′ primer to produce a population of double-stranded cDNA molecules.
2. The method of claim 1 , further comprising amplifying at least one double-stranded cDNA molecule of the population of double-stranded cDNA molecules.
3. The method of claim 2 , wherein the amplification is PCR and the PCR is performed using at least one primer selected from the group consisting of a gene-specific primer, a gene family-specific primer, a primer comprising oligo-dT, a population of random primers, and a population of degenerate primers.
4. The method of claim 2 , wherein amplification is performed using a 5′ primer comprising a nucleic acid sequence having at least 83% identity to a SL sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.
5. The method of claim 4 , wherein amplification is performed using a 5′ primer comprising a nucleic acid sequence having at least 88% identity to an SL sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.
6. The method of claim 5 , wherein amplification is performed using a 5′ primer comprising a nucleic acid sequence having at least 94% identity to an SL sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.
7. The method of claim 6 , wherein amplification is performed using a 5′ primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.
8. The method of claim 1 , wherein the cell is a heterokont cell.
9. The method of claim 8 , wherein the cell is a Eustigmatophyte cell.
10. The method of claim 9 , wherein the cell is a Nannochloropsis cell.
11. The method of claim 1 , wherein the 5′ primer has a length of from 15 to 250 nucleotides.
12. The method of claim 11 , wherein the 5′ primer has a length of from 15 to 100 nucleotides.
13. The method of claim 1 , further comprising cloning one or more of the double-stranded cDNA molecules in E. coli.
14. The method of claim 1 , further comprising sequencing at least one double-stranded cDNA molecule.
15. The method of claim 1 , wherein the 5′ primer comprises a nucleic acid sequence having at least 88% identity to a SL sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.
16. The method of claim 15 , wherein the 5′ primer comprises a nucleic acid sequence having at least 94% identity to a SL sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.
17. The method of claim 16 , wherein the 5′ primer comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.