Patent
Provided are constructs and methods for expressing a transgene in plant cells and/or plant tissues using gene regulatory elements obtained from Brassica napus.
1. A method for expressing a heterologous coding sequence in a transgenic plant, the method comprising:
transforming a plant cell with a gene expression cassette comprising a polynucleotide sequence comprising a sequence identity of at least 98% to SEQ ID NO:1 operably linked to the heterologous coding sequence, which is operably linked to a 3′-untranslated region;
isolating the transformed plant cell comprising the gene expression cassette;
regenerating the transformed plant cell into a transgenic plant; and,
obtaining the transgenic plant, wherein the transgenic plant comprises the gene expression cassette comprising the polynucleotide sequence comprising a sequence identity of at least 98% to SEQ ID NO:1.
2. The method of claim 1 , the method further comprising operably linking an intron to the polynucleotide sequence.
3. The method of claim 1 , the method further comprising operably linking a 5′-untranslated region to the polynucleotide sequence.
4. The method of claim 1 , wherein transforming the plant cell is selected from the group consisting of an Agrobacterium -mediated transformation method, a biolistics transformation method, a silicon carbide transformation method, a protoplast transformation method, and a liposome transformation method.
5. The method of claim 1 , wherein the transgenic plant is selected from the group consisting of an Arabidopsis plant, a tobacco plant, a soybean plant, a canola plant and a cotton plant.
6. The method of claim 1 , wherein the polynucleotide sequence comprises a sequence of nucleotides 1-1483 of SEQ ID NO:1.
7. A method for isolating a polynucleotide sequence comprising a sequence identity of at least 98% to SEQ ID NO:1, the method comprising:
identifying the polynucleotide sequence comprising a sequence identity of at least 98% to SEQ ID NO:1;
producing a plurality of oligonucleotide primer sequences, wherein the oligonucleotide primer sequences bind to the polynucleotide sequence comprising a sequence identity of at least 98% to SEQ ID NO:1;
amplifying the polynucleotide sequence comprising a sequence identity of at least 98% to SEQ ID NO:1 from a DNA sample with oligonucleotide primer sequences selected from the plurality of oligonucleotide primer sequences; and,
isolating the polynucleotide sequence comprising a sequence identity of at least 98% to SEQ ID NO:1.
8. The method of claim 7 , the method further comprising operably linking an intron to the polynucleotide sequence.
9. The method of claim 7 , wherein the method further comprising operably linking a 5′-untranslated region to the polynucleotide sequence.
10. The method of claim 7 , the method further comprising operably linking a transgene to the polynucleotide sequence.
11. A method for manufacturing a synthetic polynucleotide sequence comprising a sequence identity of at least 98% to SEQ ID NO:1, the method comprising: identifying the polynucleotide sequence comprising SEQ ID NO:1;
isolating the polynucleotide sequence comprising SEQ ID NO:1;
defining a plurality of polynucleotide sequences that comprise a sequence identity of at least 98% to SEQ ID NO:1;
synthesizing a polynucleotide sequence comprising a sequence identity of at least 98% to SEQ ID NO:1; and,
manufacturing a synthetic polynucleotide sequence comprising a sequence identity of at least 98% to SEQ ID NO:1.
12. The method of claim 11 , wherein the synthesizing comprises:
identifying the polynucleotide sequence comprising a sequence identity of at least 98% to SEQ ID NO:1;
producing a plurality of oligonucleotide primer sequences, wherein the oligonucleotide primer sequences bind to the polynucleotide sequence comprising a sequence identity of at least 98% to SEQ ID NO:1;
ligating the plurality of oligonucleotide primer sequences to synthesize the polynucleotide sequence comprising a sequence identity of at least 98% to SEQ ID NO:1.
13. The method of claim 12 , the method further comprising operably linking an intron to the synthetic polynucleotide sequence.
14. The method of claim 11 , the method further comprising operably linking a 5′-untranslated region to the synthetic polynucleotide sequence.
15. The method of claim 11 , the method further comprising operably linking a transgene to the synthetic polynucleotide sequence.
16. The method of claim 14 , wherein the operably linked transgene encodes a polypeptide.
17. The method of claim 11 , wherein the synthetic polynucleotide sequence comprises a sequence of nucleotides 1-1483 of SEQ ID NO:1.