Maize H3C4 promoter combined with the first intron of rice actin, chimeric gene comprising it and transformed plant

Abstract

The present invention relates to a DNA sequence, a 5′ regulatory element allowing the expression of a heterologous gene in a plant cell from a monocotyledonous plant, characterized in that it comprises, in the direction of transcription, a first DNA sequence, which is a functional fragment of the sequence of the maize H3C4 promoter, and a second DNA sequence, which is a functional fragment of the sequence of the first intron of rice actin. The invention also relates to a chimeric gene comprising the said DNA sequence and the plants transformed with the said gene.

1 - 21  (canceled)  
     
     
         22 . Fusion protein OTP/CP4.  
     
     
         23 - 35  (cancelled)  
     
     
         35 . An isolated DNA sequence comprising, in the direction of transcription, a functional fragment of the sequence of the maize H3C4 promoter and a functional fragment of the first intron of rice actin.  
     
     
         36 . The DNA sequence according to  claim 35  wherein said sequence of the maize H3C4 promoter is the AluI fragment of the maize H3C4 promoter.  
     
     
         37 . The DNA sequence according to  claim 35  wherein the functional fragment of the sequence of the maize H3C4 promoter comprises a sequence homologous to SEQ ID NO: 1.  
     
     
         38 . The DNA sequence of  claim 37  wherein said functional fragment of the first intron of rice actin comprises a sequence homologous to SEQ ID NO: 2.  
     
     
         39 . The DNA sequence of  claim 35  comprising a neutral DNA fragment between said functional fragment of the sequence of the maize H3C4 promoter and said functional fragment of the first intron of rice actin.  
     
     
         40 . An isolated DNA sequence comprising a sequence homologous to SEQ ID NO: 3.  
     
     
         41 . An expression cassette comprising a coding sequence and heterologous regulatory elements at the 5′ and 3′ positions relative to the coding sequence capable of functioning in monocotyledonous plant cells or monocotyledonous plants, wherein the 5′ regulatory element comprises the DNA sequence of  claim 35 .  
     
     
         42 . The expression cassette of  claim 41  wherein said coding sequence is a DNA sequence encoding a protein of interest.  
     
     
         43 . The expression cassette of  claim 41 , wherein the DNA sequence encoding a protein of interest is a DNA sequence encoding a selectable marker.  
     
     
         44 . The expression cassette of  claim 41  wherein said protein of interest is selected from the group consisting of a DNA sequence encoding a protein that confers herbicide tolerance, a DNA sequence encoding a protein that confers insect tolerance, and a DNA sequence encoding a protein that confers disease tolerance.  
     
     
         45 . The expression cassette of  claim 44  wherein said wherein the DNA sequence encoding a protein that confers herbicide tolerance is selected from the group consisting of a DNA sequence encoding PAT conferring tolerance to bialophos, a DNA sequence encoding an EPSPS conferring resistance to herbicides having EPSPS as a target, a DNA sequence encoding glyphosate oxidoreductase, and a DNA sequence encoding an HPPD conferring tolerance to herbicides having HPPD as a target.  
     
     
         46 . The expression cassette of  claim 45  wherein the DNA sequence encoding a protein that confers herbicide tolerance is a DNA sequence encoding an EPSPS or a DNA sequence encoding an HPPD.  
     
     
         47 . The expression cassette of  claim 46  wherein the DNA sequence encoding a protein that confers herbicide tolerance is a DNA sequence encoding CP4 or a double-mutant EPSPS.  
     
     
         48 . The expression cassette of  claim 44  wherein said DNA sequence is preceded by a sequence encoding a transit peptide.  
     
     
         49 . The expression cassette according to  claim 48  wherein said transit peptide is the optimized transit peptide.  
     
     
         50 . An expression cassette comprising, in the direction of transcription, a 5′ regulatory element of claim  1  functionally linked to a sequence encoding a fusion protein, functionally linked to a b  3 ′ l regulatory sequence, wherein said fusion protein comprises a transit peptide linked to a protein of interest.  
     
     
         51 . The expression cassette of  claim 50 , wherein said protein of interest is a protein that confers herbicide tolerance selected from the group consisting of PAT, EPSPS, glyphosate oxidoreductase, and HPPD.  
     
     
         52 . The expression cassette of  claim 51 , wherein the sequence encoding a fusion protein is a sequence encoding the fusion protein OTP/double-mutant EPSPS or a sequence encoding the fusion protein OTP/CP4.  
     
     
         53 . An isolated DNA sequence encoding a fusion protein OTP/CP4.  
     
     
         54 . An expression cassette comprising in the direction of transcription, a 5′ regulatory sequence functionally linked to a sequence encoding a fusion protein OTP/CP4, optionally linked to a 3′ regulatory sequence, wherein said expression cassette functions in plants or plant cells.  
     
     
         55 . A cloning or expression vector for the transformation of a plant cell or of a plant, which comprises an expression cassette of  claim 41  and at least one replication origin.  
     
     
         56 . The vector of  55 , wherein said vector is a plasmid.  
     
     
         57 . A method of transforming plant cells, comprising integrating the expression vector of  claim 41  into plant cells.  
     
     
         58 . A plant cell which contains at least one expression cassette of  claim 41 .  
     
     
         59 . A transformed plant which comprises a plant cell of  claim 58 .  
     
     
         60 . A transformed plant which is regenerated from a plant cell of  claim 58 .  
     
     
         61 . A transformed plant produced from the culture of a transformed plant of  claim 59  or the crossing of a transformed plant of  claim 59  with another plant.  
     
     
         62 . A transformed seed of the transformed plant of  claim 59 .  
     
     
         63 . A method of controlling weeds in a field comprising weeds and seeds or plants, said seeds or plants each comprising the expression cassette according to  claim 41 , which method comprises applying to the field a dose of herbicide which is toxic to the weeds but to which the seeds or plants are tolerant.  
     
     
         64 . A method of cultivating plants transformed with the expression cassette of  claim 41 , which method comprises, 
 sowing seeds comprising said expression cassette in a field comprising weeds;    cultivating plants from the seeds;    applying to the field a dose of herbicide which is toxic to the weeds but to which the seeds or plants are tolerant prior to sowing the seeds or during cultivation of the seeds; and;    harvesting the cultivated plants when they reach maturity.    
     
     
         65 . The method of  claim 64  wherein said herbicide is applied before sowing the seeds, before emergence of the plants or after emergence of the plants.