# US 10,415,047 B2 · TaBV transcriptional control elements, chimeric constructs and uses therefor

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- Number: US 10,415,047 B2
- Document ID: US10415047B2
- Status: Granted
- Kind: B2
- Application number: 15/194,766
- Filed: 2016-06-28
- Published: 2018-04-19
- Granted: 2019-09-17
- Earliest claimed: 2002-07-17
- Assignees: Farmacule Bioindustries Pty Ltd
- Applicants: James Langham Dale; Robert Maxwell Harding; Douglas Kenneth Becker; Gregory John Hafner; Ilin Yang
- Inventors: James Langham Dale; Robert Maxwell Harding; Douglas Kenneth Becker; Gregory John Hafner; Ilin Yang
- Web page: https://patentgrep.com/patent/US10415047B2

## CPC

- C12N 15/8216: {Methods for controlling, regulating or enhancing expression of transgenes in plant cells}
- A61P 31/12: Antivirals
- C07K 14/005: from viruses
- C12N 15/8222: {Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation}
- C12N 15/8223: {Vegetative tissue-specific promoters}
- C12N 2730/00022: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

This invention discloses a constitutive promoter from the Taro bacilliform virus (TaBV) for expression of foreign or endogenous coding sequences in plants, including dicotyledonous and monocotyledonous plants. The invention also discloses a chimeric nucleic acid construct comprising the promoter of the invention operably linked to a foreign or endogenous polynucleotide that codes for a protein of interest or a transcript capable of modulating expression of a target gene. The invention further discloses transformed plant cells, as well as differentiated plants and plant parts, containing the construct. Methods for diagnosis and treatment of viral infections, especially badnaviral infections, are also disclosed.

## Claims

### Claim 1

A chimeric DNA construct comprising a DNA molecule having promoter activity and operably linked to a foreign or endogenous DNA sequence to be transcribed, wherein the DNA molecule has at least 95% sequence identity, or hybridizes under high stringency conditions, to a biologically active fragment of the sequence set forth in SEQ ID NO:6, wherein the biologically active fragment comprises nucleotides 527 to 970 of SEQ ID NO:6, wherein the high stringency conditions comprise 1% BSA, 1 mM EDTA, 0.5 M NaHPO4 (pH 7.2), and 7% SDS for hybridization at 65° C. and washing at 68° C. in 0.2×SSC, 0.1% SDS.

### Claim 2 (depends on 1)

The construct of claim 1, further comprising a 3′ non-translated sequence that is operably linked to the foreign or endogenous DNA sequence and that functions in plant cells to terminate transcription and/or to cause addition of a polyadenylated nucleotide sequence to the 3′ end of a transcribed RNA sequence.

### Claim 3 (depends on 1)

The construct of claim 1, wherein the foreign or endogenous DNA sequence encodes a structural or regulatory protein.

### Claim 4 (depends on 1)

The construct of claim 1, wherein the foreign or endogenous DNA sequence encodes a transcript capable of modulating expression of a corresponding target gene.

### Claim 5 (depends on 4)

The construct of claim 4, wherein the transcript comprises a transcribed region aimed at downregulating the expression of the corresponding target gene.

### Claim 6 (depends on 4)

The construct of claim 4, wherein the transcript comprises a transcribed region that represents a molecule selected from the group consisting of a sense suppression molecule, an antisense RNA, a ribozyme and an RNAi molecule.

### Claim 7 (depends on 1)

The construct of claim 1, further comprising a leader sequence which modulates mRNA stability.

### Claim 8 (depends on 1)

The construct of claim 1, further comprising a targeting sequence for targeting a protein product of the foreign or endogenous DNA sequence to an intracellular compartment within plant cells or to an extracellular environment.

### Claim 9 (depends on 1)

The construct of claim 1, further comprising a selectable marker gene.

### Claim 10 (depends on 1)

The construct of claim 1, further comprising a screenable marker gene.

### Claim 11 (depends on 1)

The construct of claim 1, wherein the nucleotide sequence is constitutively expressed in a host cell.

### Claim 12 (depends on 11)

The construct of claim 11, wherein the host cell is a plant cell.

### Claim 13 (depends on 11)

The construct of claim 11, wherein the host cell is a monocotyledonous plant cell.

### Claim 14 (depends on 11)

The construct of claim 11, wherein the host cell is a non-graminaceous monocotyledonous plant cell.

### Claim 15 (depends on 11)

The construct of claim 11, wherein the host cell is a non-graminaceous monocotyledonous plant cell selected from the group consisting of Musaceae, taro, ginger, onions, garlic, pineapple, bromeliads, palms, orchids, lilies and irises.

### Claim 16 (depends on 11)

The construct of claim 11, wherein the host cell is a graminaceous monocotyledonous plant cell.

### Claim 17 (depends on 11)

The construct of claim 11, wherein the host cell is a dicotyledonous plant cell.

### Claim 18

A progeny obtained from a differentiated transgenic plant comprising a chimeric construct, wherein the progeny comprises the chimeric construct, and wherein the differentiated plant is produced by a method comprising:
(a) introducing into regenerable plant cells a chimeric DNA construct comprising a DNA molecule having promoter activity and operably linked to a foreign or endogenous DNA sequence to be transcribed, wherein the DNA molecule has at least 95% sequence identity, or hybridizes under high stringency conditions, to a biologically active fragment of the sequence set forth in SEQ ID NO:6, wherein the biologically active fragment comprises nucleotides 527 to 970 of SEQ ID NO:6, wherein the high stringency conditions comprise 1% BSA, 1 mM EDTA, 0.5 M NaHPO4 (pH 7.2), and 7% SDS for hybridization at 65° C. and washing at 68° C. in 0.2×SSC, 0.1% SDS;
(b) identifying or selecting a population of transformed plant cells; and
(c) regenerating a differentiated transgenic plant from the population.

### Claim 19

A plant part obtained from a differentiated transgenic plant produced by a method comprising the steps of:
(a) introducing into regenerable plant cells a chimeric DNA construct comprising a DNA molecule having promoter activity and operably linked to a foreign or endogenous DNA sequence to be transcribed, wherein the DNA molecule has at least 95% sequence identity, or hybridizes under high stringency conditions, to a biologically active fragment of the sequence set forth in SEQ ID NO:6, wherein the biologically active fragment comprises nucleotides 527 to 970 of SEQ ID NO:6, wherein the high stringency conditions comprise 1% BSA, 1 mM EDTA, 0.5 M NaHPO4 (pH 7.2), and 7% SDS for hybridization at 65° C. and washing at 68° C. in 0.2×SSC, 0.1% SDS;
(b) identifying or selecting a population of transformed plant cells; and
(c) regenerating a differentiated transgenic plant from the population; wherein the plant part contains the chimeric construct.

### Claim 20

A differentiated transgenic plant regenerated from transformed plant cells obtained by a method comprising the steps of:
(a) introducing into regenerable plant cells a chimeric DNA construct comprising a DNA molecule having promoter activity and operably linked to a foreign or endogenous DNA sequence to be transcribed, wherein the DNA molecule has at least 95% sequence identity, or hybridizes under high stringency conditions, to a biologically active fragment of the sequence set forth in SEQ ID NO:6, wherein the biologically active fragment comprises nucleotides 527 to 970 of SEQ ID NO:6, wherein the high stringency conditions comprise 1% BSA, 1 mM EDTA, 0.5 M NaHPO4 (pH 7.2), and 7% SDS for hybridization at 65° C. and washing at 68° C. in 0.2×SSC, 0.1% SDS;
(b) identifying or selecting a population of transformed plant cells; and
(c) regenerating a differentiated transgenic plant from the population.

### Claim 21

A transformed plant cell containing a chimeric DNA construct comprising a DNA molecule having promoter activity and operably linked to a foreign or endogenous DNA sequence to be transcribed, wherein the DNA molecule has at least 95% sequence identity, or hybridizes under high stringency conditions, to a biologically active fragment of the sequence set forth in SEQ ID NO:6, wherein the biologically active fragment comprises nucleotides 527 to 970 of SEQ ID NO:6, wherein the high stringency conditions comprise 1% BSA, 1 mM EDTA, 0.5 M NaHPO4 (pH 7.2), and 7% SDS for hybridization at 65° C. and washing at 68° C. in 0.2×SSC, 0.1% SDS.

### Claim 22

A differentiated transgenic plant comprising plant cells containing a chimeric DNA construct comprising a DNA molecule having promoter activity and operably linked to a foreign or endogenous DNA sequence to be transcribed, wherein the DNA molecule has at least 95% sequence identity, or hybridizes under high stringency conditions, to a biologically active fragment of the sequence set forth in SEQ ID NO:6, wherein the biologically active fragment comprises nucleotides 527 to 970 of SEQ ID NO:6, wherein the high stringency conditions comprise 1% BSA, 1 mM EDTA, 0.5 M NaHPO4 (pH 7.2), and 7% SDS for hybridization at 65° C. and washing at 68° C. in 0.2×SSC, 0.1% SDS.

### Claim 23 (depends on 22)

The transgenic plant of claim 22, wherein the plant is a dicotyledonous plant.

### Claim 24 (depends on 22)

The transgenic plant of claim 22, wherein the plant is a monocotyledonous plant.

### Claim 25 (depends on 22)

The transgenic plant of claim 22, wherein the plant is a graminaceous monocotyledonous plant.

### Claim 26 (depends on 22)

The transgenic plant of claim 22, wherein the plant is a non-graminaceous monocotyledonous plant.

### Claim 27 (depends on 22)

The transgenic plant of claim 22, wherein the construct comprises a selectable marker gene.

### Claim 28 (depends on 22)

The transgenic plant of claim 22, wherein the construct comprises a screenable marker gene.

### Claim 29 (depends on 22)

The transgenic plant of claim 22, wherein the expression of the chimeric DNA construct renders the differentiated transgenic plant identifiable over the corresponding non-transgenic plant.