BRASSICA NAPUS SEED SPECIFIC PROMOTERS IDENTIFIED BY MICROARRAY ANALYSIS
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:
a) transforming a plant cell with a gene expression cassette comprising a polynucleotide sequence comprising a sequence identity of at least 90% to SEQ ID NO:1 operably linked to the heterologous coding sequence, which is operably linked to a 3′-untranslated region;
b) isolating the transformed plant cell comprising the gene expression cassette;
c) regenerating the transformed plant cell into a transgenic plant; and,
d) obtaining the transgenic plant, wherein the transgenic plant comprises the gene expression cassette comprising the polynucleotide sequence comprising SEQ ID NO:1.
2 . The method of claim 1 , wherein the polynucleotide sequence comprises an intron having at least 90% sequence identity to an intron selected from the group consisting of a rice actin intron, a maize ubiquitin intron, and an Arabidopsis thaliana ubiquitin 10 intron.
3 . The method of claim 1 , wherein the polynucleotide sequence comprises a 5′-untranslated region.
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 4 , 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 . A transgenic seed from the transgenic plant of claim 1 .
7 . The method of claim 1 , wherein the polynucleotide sequence comprises a sequence of nucleotides 1-820 of SEQ ID NO:1.
8 . A method for isolating a polynucleotide sequence comprising a sequence identity of at least 90% to SEQ ID NO:1, the method comprising:
a) identifying the polynucleotide sequence comprising a sequence identity of at least 90% to SEQ ID NO:1;
b) producing a plurality of oligonucleotide primer sequences, wherein the oligonucleotide primer sequences bind to the polynucleotide sequence comprising a sequence identity of at least 90% to SEQ ID NO:1;
c) amplifying the polynucleotide sequence comprising a sequence identity of at least 90% to SEQ ID NO:1 from a DNA sample with oligonucleotide primer sequences selected from the plurality of oligonucleotide primer sequences; and,
d) isolating the polynucleotide sequence comprising a sequence identity of at least 90% to SEQ ID NO:1.
9 . The method of claim 8 , wherein the isolated polynucleotide sequence comprises an intron having at least 90% sequence identity to an intron selected from the group consisting of a rice actin intron, a maize ubiquitin intron, and an Arabidopsis thaliana ubiquitin 10 intron.
10 . The method of claim 8 , wherein the isolated polynucleotide sequence comprises a 5′-untranslated region.
11 . The method of claim 8 , wherein the isolated polynucleotide sequence is operably linked to a transgene.
12 . A gene expression cassette comprising a polynucleotide sequence with at least 90% sequence identity to SEQ ID NO:1 operably linked to a transgene, wherein the transgene is operably linked to a 3′-untranslated region.
13 . The gene expression cassette of claim 12 , wherein the transgene is selected from the group consisting of insecticidal resistance coding sequences, herbicide tolerance coding sequences, nitrogen use efficiency coding sequences, water use efficiency coding sequences, nutritional quality coding sequences, DNA binding coding sequences, and selectable marker coding sequences.
14 . The gene expression cassette of claim 12 , wherein the 3′-untranslated region has at least 90% sequence identity to SEQ ID NO:2.
15 . A recombinant vector comprising the gene expression cassette of claim 12 .
16 . A transgenic cell comprising the gene expression cassette of claim 12 .
17 . A transgenic plant comprising the transgenic cell of claim 16 .
18 . The transgenic plant of claim 17 , wherein the plant is selected from the group consisting of an Arabidopsis plant, a tobacco plant, a soybean plant, a canola plant and a cotton plant.
19 . A transgenic seed from the transgenic plant of claim 17 .
20 . The method of claim 12 , wherein the isolated polynucleotide sequence comprises a sequence of nucleotides 1-820 of SEQ ID NO:1.
21 . A method for manufacturing a synthetic polynucleotide sequence comprising a sequence identity of at least 90% to SEQ ID NO:1, the method comprising:
a) identifying the polynucleotide sequence comprising SEQ ID NO:1;
b) isolating the polynucleotide sequence comprising SEQ ID NO:1;
c) defining a plurality of polynucleotide sequences that comprise a sequence identity of at least 90% to SEQ ID NO:1;
d) synthesizing a polynucleotide sequence comprising a sequence identity of at least 90% to SEQ ID NO:1; and,
e) manufacturing a synthetic polynucleotide sequence comprising a sequence identity of at least 90% to SEQ ID NO:1.
22 . The method of claim 21 , wherein the synthesizing comprises:
a) identifying the polynucleotide sequence comprising a sequence identity of at least 90% to SEQ ID NO:1;
b) producing a plurality of oligonucleotide primer sequences, wherein the oligonucleotide primer sequences bind to the polynucleotide sequence comprising a sequence identity of at least 90% to SEQ ID NO:1;
c) ligating the plurality of oligonucleotide primer sequences to synthesize the polynucleotide sequence comprising a sequence identity of at least 90% to SEQ ID NO:1.
23 . The method of claim 21 , wherein the synthetic polynucleotide sequence comprises an intron having at least 90% sequence identity to an intron selected from the group consisting of a rice actin intron, a maize ubiquitin intron, and an Arabidopsis thaliana ubiquitin 10 intron.
24 . The method of claim 21 , wherein the synthetic polynucleotide sequence comprises a 5′-untranslated region.
25 . The method of claim 21 , wherein the synthesized polynucleotide sequence is operably linked to a transgene.
26 . The method of claim 24 , wherein the operably linked transgene encodes a polypeptide.
27 . A gene expression cassette comprising the synthesized polynucleotide sequence comprising a sequence identity of at least 90% to SEQ ID NO:1 operably linked to the transgene of claim 93 , that is operably linked to a 3′-untranslated region.
28 . The gene expression cassette of claim 27 , wherein the 3′-untranslated region has at least 90% sequence identity to SEQ ID NO:2.
29 . A recombinant vector comprising the gene expression cassette of claim 27 .
30 . A transgenic cell comprising the gene expression cassette of claim 27 .
31 . A transgenic plant comprising the transgenic cell of claim 30 .
32 . The transgenic plant of claim 31 , wherein the dicotyledonous plant is selected from the group consisting of an Arabidopsis plant, a tobacco plant, a soybean plant, a canola plant and a cotton plant.
33 . A transgenic seed from the transgenic plant of claim 31 .
34 . The method of claim 21 , wherein the synthetic polynucleotide sequence comprises a sequence of nucleotides 1-820 of SEQ ID NO:1.