# US 10,870,859 B2 · U6 polymerase III promoter and methods of use

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- Number: US 10,870,859 B2
- Document ID: US10870859B2
- Status: Granted
- Kind: B2
- Application number: 16/674,698
- Filed: 2019-11-05
- Published: 2020-02-27
- Granted: 2020-12-22
- Earliest claimed: 2013-08-22
- Assignees: E. I. Du Pont de Nemours and Company
- Applicants: E. I. Du Pont De Nemours and Company
- Inventors: Zhongsen Li; Zhan-Bin Liu
- Web page: https://patentgrep.com/patent/US10870859B2

## CPC

- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C12N 15/8216: {Methods for controlling, regulating or enhancing expression of transgenes in plant cells}
- C12N 9/222: {Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes}
- C12N 15/00: Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor (mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00)
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N 15/8205: {Agrobacterium mediated transformation}
- C12N 15/8207: {by mechanical means, e.g. microinjection, particle bombardment, silicon whiskers}
- C12N 15/8213: {Targeted insertion of genes into the plant genome by homologous recombination}
- C12N 15/8241: {Phenotypically and genetically modified plants via recombinant DNA technology}
- C12N 15/8247: {involving modified lipid metabolism, e.g. seed oil composition}
- C12N 15/8262: {involving plant development}
- C12N 15/8274: {for herbicide resistance}
- A01H 1/00: Processes for modifying genotypes {; Plants characterised by associated natural traits} (A01H4/00 takes precedence)
- C12N 2310/10: Type of nucleic acid
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2310/315: Phosphorothioates
- C12N 2310/3231: having an additional ring, e.g. LNA, ENA
- C12N 2310/3341: 5-Methylcytosine
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A01H: NEW PLANTS OR {NON-TRANSGENIC} PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES

## Abstract

The disclosure relates to gene expression regulatory sequences from maize, specifically to the promoter of a maize U6 polymerase III gene and fragments thereof and their use in promoting the expression of one or more heterologous nucleic acid fragments in plants. The invention further discloses compositions, polynucleotide constructs, transformed host cells, mutated plants, transgenic plants and seeds containing the recombinant construct with the promoter, and methods for preparing and using the same.

## Claims

### Claim 1

A recombinant DNA construct comprising a promoter nucleotide sequence comprising a region of homology of at least 98% sequence identity with at least 100 contiguous nucleotides of SEQ ID NO: 9, operably linked to at least one heterologous sequence, wherein said nucleotide sequence controls the expression of the heterologous sequence, wherein the at least 100 contiguous nucleotides comprise a TATA box, and wherein the nucleotide sequence comprises said TATA box.

### Claim 2 (depends on 1)

The recombinant DNA construct of claim 1, wherein the nucleotide sequence comprises at least 100 contiguous nucleotides of SEQ ID NO: 9, wherein the at least 100 contiguous nucleotides comprise a TATA box, and wherein the nucleotide sequence comprises said TATA box.

### Claim 3 (depends on 1)

A vector comprising the recombinant DNA construct of claim 1.

### Claim 4 (depends on 1)

A cell comprising the recombinant DNA construct of claim 1.

### Claim 5 (depends on 4)

The cell of claim 4, wherein the cell is a plant cell.

### Claim 6 (depends on 1)

A transgenic plant comprising the recombinant DNA construct of claim 1.

### Claim 7 (depends on 6)

The transgenic plant of claim 6, wherein said plant is a monocot plant.

### Claim 8 (depends on 7)

The transgenic plant of claim 7, wherein the plant is a maize plant.

### Claim 9 (depends on 7)

A transgenic seed produced by the transgenic plant of claim 7, wherein the transgenic seed comprises the recombinant DNA construct.

### Claim 10 (depends on 1)

The recombinant DNA construct of claim 1, wherein the at least one heterologous sequence is selected from the group consisting of: a reporter coding sequence, a selection marker, a functional RNA, a disease resistance conferring coding sequence, a herbicide resistance conferring coding sequence, an insect resistance conferring coding sequence, a carbohydrate metabolism coding sequence, a fatty acid metabolism coding sequence, an amino acid metabolism coding sequence, a drought resistance coding sequence, a cold resistance coding sequence, a heat resistance coding sequence, and a salt resistance coding sequence.

### Claim 11 (depends on 1)

The recombinant DNA construct of claim 1, wherein the at least one heterologous sequence encodes a sequence selected from the group consisting of: a reporter protein, a selection marker, a functional RNA, a protein conferring disease resistance, a protein conferring herbicide resistance, a protein conferring insect resistance, a carbohydrate metabolism protein, a fatty acid metabolism protein, an amino acid metabolism protein, a drought resistance protein, a cold resistance protein, a heat resistance protein, and a salt resistance protein.

### Claim 12

A method of expressing a coding sequence or a functional RNA in a plant comprising:
(a) introducing a recombinant DNA construct into the plant wherein the recombinant DNA construct comprises a promoter nucleotide sequence comprising a region of homology of at least 98% sequence identity with at least 100 contiguous nucleotides of SEQ ID NO: 9, operably linked to at least one heterologous sequence, wherein said nucleotide sequence controls the expression of the heterologous sequence, wherein the at least 100 contiguous nucleotides comprise a TATA box, wherein the promoter nucleotide sequence comprises said TATA box, and wherein the at least one heterologous sequence comprises a coding sequence or encodes a functional RNA;
(b) growing the plant of step (a); and
(c) selecting a plant displaying expression of the coding sequence or the functional RNA of the recombinant DNA construct.

### Claim 13

A method of transgenically altering a plant trait, comprising:
(a) introducing a recombinant DNA construct into the plant, wherein the recombinant DNA construct comprises a promoter nucleotide sequence comprising a region of homology of at least 98% sequence identity with at least 100 contiguous nucleotides of SEQ ID NO: 9, operably linked to at least one heterologous sequence, wherein said promoter nucleotide sequence controls the expression of the heterologous sequence, wherein the at least 100 contiguous nucleotides comprise a TATA box, and wherein the promoter nucleotide sequence comprises said TATA box;
(b) growing a fertile, mature plant resulting from step (a); and
(c) selecting a plant expressing the at least one heterologous sequence in at least one plant tissue having the altered trait.

### Claim 14 (depends on 13)

The method of claim 13, wherein the trait is selected from the group consisting of disease resistance, herbicide resistance, insect resistance carbohydrate metabolism, fatty acid metabolism, amino acid metabolism, drought resistance, cold resistance, heat resistance, and salt resistance.

### Claim 15

A method for increasing expression of at least one heterologous sequence in a plant comprising:
(a) transforming a plant cell with a recombinant DNA construct, wherein the recombinant DNA construct comprises a promoter nucleotide sequence comprising a region of homology of at least 98% sequence identity with at least 100 contiguous nucleotides of SEQ ID NO: 9, operably linked to at least one heterologous sequence, wherein said promoter nucleotide sequence controls the expression of the heterologous sequence, wherein the at least 100 contiguous nucleotides comprise a TATA box, wherein the promoter nucleotide sequence comprises said TATA box;
(b) growing fertile mature plants from transformed plant cell of step (a); and
(c) selecting plants containing the transformed plant cell wherein the expression of the heterologous sequence is increased.

### Claim 16 (depends on 15)

The method of claim 15 wherein the plant is a maize plant.

### Claim 17

A plant transformed with a recombinant DNA construct comprising a promoter and a heterologous nucleic acid fragment operably linked to said promoter, wherein said promoter controls expression of said heterologous nucleic acid fragment in a plant cell, and further wherein said promoter comprises a region of homology of at least 98% sequence identity with at least 100 contiguous nucleotides SEQ ID NO: 9; wherein the at least 100 contiguous nucleotides comprise a TATA box, and wherein the promoter comprises said TATA box.

### Claim 18

A recombinant DNA construct comprising a promoter comprising a nucleotide sequence comprising a region of homology of at least 98% sequence identity with at least 100 contiguous nucleotides with SEQ ID NO: 9, wherein the at least 100 contiguous nucleotides comprise a TATA box, wherein the nucleotide sequence comprises said TATA box, wherein said promoter is operably linked to a heterologous nucleic acid fragment, and wherein said heterologous nucleic acid fragment expresses a guide RNA.