US2009031440A1PendingUtilityA1
Expression Cassettes for Seed-Preferential Expression in Plants
Est. expiryFeb 26, 2025(expired)· nominal 20-yr term from priority
C12N 15/8234
52
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Claims
Abstract
The present invention relates to expression cassettes comprising transcription regulating nucleotide sequences with seed-preferential or seed-specific expression profiles in plants obtainable from the Arabidopsis thaliana gene described by the locus At1g48130 or its orthologs from other plant species (such as Linum usitatissimum ). The transcription regulating nucleotide sequences preferably exhibit strong expression activity especially in seeds and seed tissues.
Claims
exact text as granted — not AI-modified1 . An expression cassette for regulating seed-specific or seed-preferential expression in plants comprising
a) at least one transcription regulating nucleotide sequence of a gene encoding a peroxiredoxin (PER1) protein, and functionally linked thereto b) at least one nucleic acid sequence which is heterologous in relation to said transcription regulating nucleotide sequence.
2 . An expression cassette for regulating seed-specific or seed-preferential expression in plants comprising
a) at least one transcription regulating nucleotide sequence of a plant gene, said plant gene is selected from the group consisting of
i) the gene described by the GenBank Arabidopsis thaliana genome loci At1g48130, and
ii) orthologous genes of the gene described by the GenBank Arabidopsis thaliana genome loci At1g48130,
and functionally linked thereto
b) at least one nucleic acid sequence which is heterologous in relation to said transcription regulating nucleotide sequence.
3 . The expression cassette of claim 1 , wherein the transcription regulating nucleotide sequence is obtainable from plant genomic DNA from a gene encoding a polypeptide which
a) comprises at least one sequence motive selected from the group consisting of the amino acid sequences
i)
GDTVPNL,
(SEQ ID NO: 37)
ii)
GDFTPVC,
(SEQ ID NO: 39)
iii)
VKLLGLS,
(SEQ ID NO: 41)
iv)
YPI(I/M)ADP,
(SEQ ID NO: 43)
v)
KLSFLYP,
(SEQ ID NO: 45)
vi)
GRNMDEV,
(SEQ ID NO: 47)
vii)
(I/V)ATP(V/A)NW,
(SEQ ID NO: 49)
and
viii)
PSKKGYL,
(SEQ ID NO: 51)
or
b) has at least 50% amino acid sequence identity to a polypeptide selected from the group described by SEQ ID NO: 13, 15, 17, 19, 21, and 23.
4 . The expression cassette of claim 1 , wherein the transcription regulating nucleotide sequence is obtainable from plant genomic DNA from a gene encoding a polypeptide which
a) comprises at least one sequence motive selected from the group consisting of the amino acid sequences
i)
GDTVPNL,
(SEQ ID NO: 37)
ii)
GDFTPVC,
(SEQ ID NO: 39)
iii)
VKLLGLS,
(SEQ ID NO: 41)
iv)
YPI(I/M)ADP,
(SEQ ID NO: 43)
v)
KLSFLYP,
(SEQ ID NO: 45)
vi)
GRNMDEV,
(SEQ ID NO: 47)
vii)
(I/V)ATP(V/A)NW,
(SEQ ID NO: 49)
and
viii)
PSKKGYL,
(SEQ ID NO: 51)
and
b) has at least 50% amino acid sequence identity to a polypeptide selected from the group described by SEQ ID NO: 13, 15, 17, 19, 21, and 23.
5 . The expression cassette of claim 1 , wherein the transcription regulating nucleotide sequence comprises at least one sequence region selected from the group consisting of
a) a region comprising at least two of the promoter motifs described by SEQ ID NO: 53, 54, 55, 56, 57, 58, or 59, b) a region comprising at least two of the promoter motifs described by SEQ ID NO: 60, 61, 62, 63 or 64, and c) a region comprising at least two of the promoter motifs described by SEQ ID NO: 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, or 76.
6 . The expression cassette of claim 5 , wherein the transcription regulating nucleotide sequence comprises at least one sequence region selected from the group consisting of
a) a region comprising at least two of the promoter motifs described by SEQ ID NO: 77, 78, 79, 80, 81, 82, or 83, b) a region comprising at least two of the promoter motifs described by SEQ ID NO: 84, 85, 86, 87, or 88, and c) a region comprising at least two of the promoter motifs described by SEQ ID NO: 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.
7 . The expression cassette of claim 5 , wherein the transcription regulating nucleotide sequence comprises at least one sequence region selected from the group consisting of
a) a region comprising at least two of the promoter motifs described by SEQ ID NO: 101, 102, 103, 104, 105, 106, or 107, b) a region comprising at least two of the promoter motifs described by SEQ ID NO: 108, 109, 110, 111, or 112, and c) a region comprising at least two of the promoter motifs described by SEQ ID NO: 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, or 124.
8 . The expression cassette of claim 1 , wherein the transcription regulating nucleotide sequence is selected from the group of sequences consisting of
i) the sequence described by SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, ii) a fragment of at least 50 consecutive bases of a sequence under i), iii) a nucleotide sequence having substantial similarity with a sequence identity of at least 50% to a transcription regulating nucleotide sequence described by SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11; iv) a nucleotide sequence capable of hybridizing under conditions equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50° C. with washing in 2×SSC, 0.1% SDS at 50° C. to a transcription regulating nucleotide sequence described by SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, or the complement thereof; v) a nucleotide sequence capable of hybridizing under conditions equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50° C. with washing in 2×SSC, 0.1% SDS at 50° C. to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of a transcription regulating nucleotide sequence described by SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, or the complement thereof; and vi) a nucleotide sequence which is the complement or reverse complement of any of the previously mentioned nucleotide sequences under i) to v).
9 . The expression cassette of claim 8 , wherein the sequences specified under ii), iii), iv) v) and vi) are capable to modify transcription in a plant cell or organism.
10 . The expression cassette of claim 8 , wherein the sequences specified under ii), iii), iv) v) and vi) have substantially the same transcription regulating activity as the transcription regulating nucleotide sequence described by SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.
11 . The expression cassette of claim 8 , wherein the sequences specified under iii) have a sequence identity of at least 80% to the sequence described by SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.
12 . The expression cassette of claim 8 , wherein the sequences specified under iv) or v) are hybridizing under stringent conditions with the specified target sequence.
13 . The expression cassette of claim 1 , wherein expression of the nucleic acid sequence results in expression of a protein, or expression of a antisense RNA, sense or double-stranded RNA.
14 . The expression cassette of claim 1 , wherein expression of the nucleic acid sequence confers to the plant an agronomically valuable trait.
15 . The expression cassette of claim 1 , wherein the expression cassette further comprises functionally liked to the nucleotide sequence to be expressed at least one sequence selected from the group consisting of:
a) the sequences described by SEQ ID NO: 125, 126, and 127, and b) a sequence comprising a fragment of at least 25 consecutive nucleotides of a sequence described by SEQ ID NO: 125, 126, or 127, c) a sequence having an identity of at least 50% to a sequence described by SEQ ID NO: 125, 126, or 127, and d) a sequence capable of hybridizing under conditions equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50° C. with washing in 2×SSC, 0.1% SDS at 50° C. to a nucleic acid comprising 50 to 200 or more (preferably all) consecutive nucleotides of as sequence described by SEQ ID NO: 125, 126, or 127, or the complement thereof,
wherein said sequence is capable of mediating RNA stabilization, stabilization of RNA expression, RNA transcription termination and/or RNA polyadenylation.
16 . An isolated nucleotide sequence selected from the group of sequences consisting of:
a) the sequence as described by SEQ ID NO: 1, 2, 3, or 4, and sequences having an identity of at least 99% to a sequence as described by SEQ ID NO: 1, 2, 3, or 4, and sequences comprising at least 600 consecutive nucleotides of a sequence as described by SEQ ID NO: 1, 2, 3, or 4, and b) the sequence as described by SEQ ID NO: 9, 10, or 11, and sequences having an identity of at least 40% to a sequence as described by SEQ ID NO: 5 or 26, and sequences comprising at least 15 consecutive nucleotides of a sequence as described by SEQ ID NO: 9, 10, or 11.
17 . A vector comprising the isolated nucleic acid sequence of claim 16 .
18 . A transgenic host cell or non-human organism comprising the vector of claim 17 .
19 . A transgenic plant or plant cell comprising the vector of claim 17 .
20 . The transgenic plant or plant cell of claim 19 , wherein said plant or plant cell is from a plant used for oil production.
21 . A method for identifying and/or isolating a late or mature seed specific or preferential transcription regulating nucleotide sequence, wherein the method comprises utilizing a nucleic acid sequence encoding a polypeptide as described by SEQ ID NO: 13, 15, 17, 19, 21, or 23, or a part of at least 15 consecutive nucleotides of said nucleic acid sequence for said identification and/or isolation.
22 . The method of claim 21 , wherein the nucleic acid sequences is described by SEQ ID NO: 12, 14, 16, 18, 20, or 22 or a part of at least 15 consecutive nucleotides thereof.
23 . The method of claim 21 , wherein said identification and/or isolation is realized by a method selected from polymerase chain reaction, hybridization, and database screening.
24 . A method for providing or producing a transgenic expression cassette for heterologous expression in late or mature seeds of plants comprising the steps of:
I. isolating a transcription regulating nucleotide sequence of a plant gene utilizing at least one nucleic acid sequence encoding a polypeptide described by SEQ ID NO: 13, 15, 17, 19, 21, or 23, or a part of at least 15 consecutive nucleotides of said nucleic acid sequence, and II. functionally linking said transcription regulating nucleotide sequence to another nucleotide sequence of interest, which is heterologous in relation to said transcription regulating nucleotide sequence.
25 . A method for providing a transgenic expression cassette for late or mature seed-specific or late or mature seed-preferential expression comprising the steps of:
I. isolating a seed-preferential or seed-specific transcription regulating nucleotide sequence utilizing at least one nucleic acid sequence or a part thereof, wherein said sequence encodes a polypeptide described by SEQ ID NO: 13, 15, 17, 19, 21, or 23, or a part of at least 15 consecutive nucleotides thereof, and II. functionally linking said seed-preferential or seed-specific transcription regulating nucleotide sequence to another nucleotide sequence of interest, which is heterologous in relation to said seed-preferential or seed-specific transcription regulating nucleotide sequence.
26 . The method of claim 21 , wherein the nucleotide sequence utilized for isolation of said transcription regulating nucleotide sequence encodes a polypeptide comprising at least one sequence motive selected from the group consisting of the amino acid sequences
i)
GDTVPNL,
(SEQ ID NO: 37)
ii)
GDFTPVC,
(SEQ ID NO: 39)
iii)
VKLLGLS,
(SEQ ID NO: 41)
iv)
YPI(I/M)ADP,
(SEQ ID NO: 43)
v)
KLSFLYP,
(SEQ ID NO: 45)
vi)
GRINMDEV,
(SEQ ID NO: 47)
vii)
(I/V)ATP(V/A)NW,
(SEQ ID NO: 49)
and
viii)
PSKKGYL.
(SEQ ID NO: 51)
27 . A synthetic transcription regulating sequence comprising at least one sequence region selected from the group consisting of
a) a region comprising at least two of the promoter motifs described by SEQ ID NO: 53, 54, 55, 56, 57, 58, or 59, b) a region comprising at least two of the promoter motifs described by SEQ ID NO: 60, 61, 62, 63 or 64, and c) a region comprising at least two of the promoter motifs described by SEQ ID NO: 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, or 76.
28 . The synthetic transcription regulating sequence of claim 27 , wherein said sequence comprises at least one sequence region selected from the group consisting of
a) a region comprising at least two of the promoter motifs described by SEQ ID NO: 77, 78, 79, 80, 81, 82, or 83, b) a region comprising at least two of the promoter motifs described by SEQ ID NO: 84, 85, 86, 87, or 88, and c) a region comprising at least two of the promoter motifs described by SEQ ID NO: 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.
29 . The synthetic transcription regulating sequence of claim 27 , wherein said sequence comprises at least one sequence region selected from the group consisting of
a) a region comprising at least two of the promoter motifs described by SEQ ID NO: 101, 102, 103, 104, 105, 106, or 107, b) a region comprising at least two of the promoter motifs described by SEQ ID NO: 108, 109, 110, 111, or 112, and c) a region comprising at least two of the promoter motifs described by SEQ ID NO: 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, or 124.
30 . A method for providing a synthetic transcription regulating nucleotide sequence for transcription in late or mature seed specific or late or mature seed preferential, wherein the method comprises combining isolated promoter motifs or cluster of promoter motifs, wherein said motifs or clusters of motifs comprise at least 5 of the motifs described by any of SEQ ID NO: 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, or 76.
31 . The method of claim 30 , wherein the isolated promoter motifs or cluster of promoter motifs comprise at least 5 of the motifs described by any of SEQ ID NO: 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.
32 . The method of claim 30 , wherein the isolated promoter motifs or cluster of promoter motifs comprise at least 5 of the motifs described by any of SEQ ID NO: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, or 124.
33 . An expression cassette comprising
i) at least one transcription regulating nucleotide sequence capable to mediate transcription in plants and functionally linked thereto ii) at least one nucleic acid sequence, and functionally linked thereto iii) at least one sequence selected from the group consisting of:
a) the sequences described by SEQ ID NO: 125, 126, or 127,
b) a sequence comprising a fragment of at least 25 consecutive nucleotides of a sequence described by SEQ ID NO: 125, 126, or 127,
c) a sequence having an identity of at least 50% to a sequence described by SEQ ID NO: 125, 126, or 127 and
d) a sequence capable of hybridizing under conditions equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50° C. with washing in 2×SSC, 0.1% SDS at 50° C. to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of the sequence described by SEQ ID NO: 125, 126, or 127, or the complement thereof,
wherein said sequence iii) is capable of mediating RNA stabilization, stabilization of RNA expression, RNA transcription termination and/or RNA polyadenylation; and
wherein either said transcription regulating sequence i) or sequence iii) or both i) and iii) are heterologous in relation to said nucleic acid sequence ii).
34 . (canceled)
35 . The method of claim 36 , wherein the transcription regulating nucleotide sequence is selected from the group of sequences consisting of
i) the sequence described by SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, ii) a fragment of at least 50 consecutive bases of a sequence under i), iii) a nucleotide sequence having a sequence identity of at least 50% to a transcription regulating nucleotide sequence described by SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11; iv) a nucleotide sequence capable of hybridizing under conditions equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50° C. with washing in 2×SSC, 0.1% SDS at 50° C. to a transcription regulating nucleotide sequence described by SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, or the complement thereof; v) a nucleotide sequence capable of hybridizing under conditions equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50° C. with washing in 2×SSC, 0.1% SDS at 50° C. to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of a transcription regulating nucleotide sequence described by SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, or the complement thereof.
36 . A method for late or mature seed-specific or preferential transcription of at least one nucleic acid molecule, comprising:
a) functionally linking at least one transcription regulating nucleotide sequence of the gene described as peroxiredoxin (PER1) or as described by the GenBank Arabidopsis thaliana genome loci At1g48130, to at least one nucleic acid sequence which is heterologous in relation to said transcription regulating nucleotide sequence; b) introducing the nucleic acid molecule into a plant cell and producing seeds.
37 . A vector comprising the expression cassette of claim 1 .
38 . A transgenic host cell or non-human organism comprising the expression cassette of claim 1 .
39 . A transgenic plant or plant cell comprising the expression cassette of claim 1 .
40 . The transgenic plant or plant cell of claim 39 , wherein said plant or plant cell is from a plant used for oil production.Join the waitlist — get patent alerts
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