# US 9,752,155 B2 · Constructs for expressing transgenes using regulatory elements from brachypodium ubiquitin 1C genes

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- Number: US 9,752,155 B2
- Document ID: US9752155B2
- Status: Granted
- Kind: B2
- Application number: 14/472,977
- Filed: 2014-08-29
- Published: 2015-03-05
- Granted: 2017-09-05
- Earliest claimed: 2013-08-30
- Assignees: Dow AgroSciences LLC
- Applicants: Dow AgroSciences, LLC
- Inventors: Sandeep Kumar; Manju Gupta; Diaa Alabed
- Web page: https://patentgrep.com/patent/US9752155B2

## CPC

- C12N 15/8216: {Methods for controlling, regulating or enhancing expression of transgenes in plant cells}
- C07K 14/415: from plants
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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

Provided are constructs and methods for expressing a transgene in plant cells and/or plant tissues using the regulatory elements, including the promoters and/or 3′-UTRs, isolated from Brachypodium distachyon ubiquitin genes.

## Claims

### Claim 1

A nucleic acid vector comprising a promoter, wherein
i) said promoter is operably linked to a polylinker sequence;
ii) said promoter is operably linked to a non-ubiquitin-coding sequence; or
iii) said promoter is operably linked to a sequence comprising a polylinker sequence and a non-ubiquitin-coding sequence, wherein said promoter comprises SEQ ID NO: 1 or a sequence that has at least 99% sequence identity with SEQ ID NO: 1 and can initiate transcription of an operably linked coding sequence.

### Claim 2 (depends on 1)

The nucleic acid vector of claim 1 wherein said promoter is less than 3 kb in length.

### Claim 3 (depends on 1)

The nucleic acid vector of claim 1 wherein said promoter consists of SEQ ID NO: 1.

### Claim 4 (depends on 1)

The nucleic acid vector of claim 1 further comprising a sequence encoding a selectable marker.

### Claim 5 (depends on 4)

The nucleic acid vector of claim 4 wherein said promoter is operably linked to a transgene.

### Claim 6 (depends on 5)

The nucleic acid vector of claim 5 wherein the transgene encodes a selectable marker or a gene product conferring insecticidal resistance, herbicide tolerance, nitrogen use efficiency, water use efficiency, or nutritional quality.

### Claim 7 (depends on 1)

The nucleic acid vector of claim 1 further comprising a 3′ untranslated sequence comprising SEQ ID NO: 4 or a sequence that has at least 99% sequence identity with SEQ ID NO: 4, wherein the 3′ untranslated sequence is operably linked to said polylinker or said coding sequence.

### Claim 8 (depends on 1)

The nucleic acid vector of claim 1 further comprising a 5′ untranslated sequence comprising SEQ ID NO: 11 or a sequence that has at least 99% sequence identity with SEQ ID NO: 11, wherein the 5′ untranslated sequence is inserted between, and operably linked to, said promoter sequence and said polylinker or said coding sequence.

### Claim 9 (depends on 8)

The nucleic acid vector of claim 8 further comprising an intron sequence inserted after the 5′ untranslated sequence.

### Claim 10 (depends on 9)

The nucleic acid vector of claim 9 wherein the intron sequence comprises SEQ ID NO: 7.

### Claim 11 (depends on 1)

The nucleic acid vector of claim 1 wherein the promoter consists of SEQ ID NO: 40 or a sequence having at least 99% sequence identity with SEQ ID NO: 40, and said promoter being operably linked to said polylinker or said coding sequence, wherein said promoter can initiate transcription of the operably linked coding sequence.

### Claim 12 (depends on 1)

The nucleic acid vector of claim 1 wherein the promoter consists of SEQ ID NO: 15 or a sequence having at least 99% sequence identity with SEQ ID NO: 15, said promoter being operably linked to said polylinker or said coding sequence, wherein said promoter can initiate transcription of the operably linked coding sequence.

### Claim 13 (depends on 12)

The nucleic acid vector of claim 12 further comprising a 3′ untranslated sequence comprising SEQ ID NO: 4 or a sequence that has at least 99% sequence identity with SEQ ID NO: 4, wherein the 3′ untranslated sequence is operably linked to said polylinker or said coding sequence.

### Claim 14

A non-Brachypodium plant comprising a Brachypodium promoter, wherein said Brachypodium promoter comprises SEQ ID NO: 1, or a sequence that has at least 99% sequence identity with SEQ ID NO: 1 operably linked to a coding sequence wherein said Brachypodium promoter can initiate transcription of the operably linked coding sequence.

### Claim 15 (depends on 14)

The plant of claim 14 wherein said plant is selected from the group consisting of maize, wheat, rice, sorghum, oats, rye, bananas, sugar cane, soybean, cotton, sunflower, and canola.

### Claim 16 (depends on 14)

The plant of claim 14 wherein said plant is Zea mays.

### Claim 17 (depends on 16)

The plant of claim 16 wherein the operably linked coding sequence is a transgene, further wherein said transgene is inserted into the genome of said plant.

### Claim 18 (depends on 14)

The plant of claim 14 further comprising a 5′ untranslated sequence comprising SEQ ID NO: 11 or a sequence that has at least 99% sequence identity with SEQ ID NO: 11, wherein the 5′ untranslated sequence is inserted between, and operably linked to, said Brachypodium promoter and said coding sequence.

### Claim 19 (depends on 18)

The plant of claim 18 further comprising an intron sequence inserted after the 5′ untranslated sequence.

### Claim 20 (depends on 19)

The plant of claim 19 wherein the intron sequence comprises SEQ ID NO: 7.

### Claim 21 (depends on 18)

The plant of claim 18 further comprising a 3′ untranslated sequence comprising SEQ ID NO: 4 or a sequence that has at least 99% sequence identity with SEQ ID NO: 4, wherein the 3′ untranslated sequence is operably linked to said coding sequence.

### Claim 22 (depends on 14)

The plant of claim 14 wherein the Brachypodium promoter consists of SEQ ID NO: 40 or a sequence having at least 99% sequence identity with SEQ ID NO: 40 and can initiate transcription of the operably linked coding sequence further wherein said Brachypodium promoter is operably linked to said coding sequence.

### Claim 23 (depends on 14)

The plant of claim 14 wherein the Brachypodium promoter consists of SEQ ID NO: 15 or a sequence having at least 99% sequence identity with SEQ ID NO: 15 and can initiate transcription of an operably linked coding sequence further wherein said Brachypodium promoter is operably linked to said coding sequence.

### Claim 24 (depends on 23)

The plant of claim 23 further comprising a 3′ untranslated sequence comprising SEQ ID NO: 4 or a sequence that has at least 99% sequence identity with SEQ ID NO: 4, wherein the 3′ untranslated sequence is operably linked to said coding sequence.

### Claim 25

A nucleic acid vector comprising a transcription terminator, wherein
i) said terminator is operably linked to a polylinker sequence;
ii) said terminator is operably linked to a non-ubiquitin-coding sequence; or
iii) said terminator is operably linked to a sequence comprising a polylinker sequence and a non-ubiquitin-coding sequence, wherein said transcription terminator comprises SEQ ID NO: 4 or a sequence that has at least 99% sequence identity with SEQ ID NO: 4.

### Claim 26 (depends on 25)

The nucleic acid vector of claim 25 wherein said transcription terminator is less than 1 kb in length.

### Claim 27 (depends on 26)

The nucleic acid vector of claim 26 wherein said transcription terminator consists of SEQ ID NO: 4.