Compositions and method for modulation of gene expression in plants

Abstract

An enzymatic nucleic acid molecule with RNA cleaving activity, wherein said nucleic acid molecule modulates the expression of an gene in a plant. A transgenic plant comprising nucleic acids encoding for an enzymatic nucleic acid molecule with RNA cleaving activity, wherein said nucleic acid molecule modulates the expression of a gene in said plant.

1 . An enzymatic nucleic acid molecule with RNA cleaving activity, wherein said nucleic acid molecule modulates the expression of a plant gene.  
     
     
         2 . The enzymatic nucleic acid molecule of  claim 1 , wherein said plant is a monocotyledon.  
     
     
         3 . The enzymatic nucleic acid molecule of  claim 1 , wherein said plant is a dicotyledon.  
     
     
         4 . The enzymatic nucleic acid molecule of  claim 1 , wherein said plant is a gymnosperm.  
     
     
         5 . The enzymatic nucleic acid molecule of  claim 1 , wherein said plant is an angiosperm.  
     
     
         6 . The enzymatic nucleic acid molecule of  claim 1 , wherein said nucleic acid is in a hammerhead configuration.  
     
     
         7 . The enzymatic nucleic acid molecule of  claim 1 , wherein said nucleic acid is in a hairpin configuration.  
     
     
         8 . The enzymatic nucleic acid molecule of  claim 1 , wherein said nucleic acid is in a hepatitis Δ virus, group I intron, group II intron, VS nucleic acid or RNaseP nucleic acid configuration.  
     
     
         9 . The enzymatic nucleic acid of any of claims  1 - 8 , wherein said nucleic acid comprises between 12 and 100 bases complementary to RNA of said gene.  
     
     
         10 . The enzymatic nucleic acid of any of claims  1 - 8 , wherein said nucleic acid comprises between 14 and 24 bases complementary to RNA of said gene.  
     
     
         11 . The enzymatic nucleic acid of  claim 6 , wherein said hammerhead comprises a stem II region of length greater than on equal to two base-pairs.  
     
     
         12 . The enzymatic nucleic acid of  claim 7 , wherein said hairpin comprises a stem II region of length between three and seven base-pairs.  
     
     
         13 . The enzymatic nucleic acid of  claim 7 , wherein said hairpin comprises a stem IV region of length greater than or equal to two base-pairs.  
     
     
         14 . The enzymatic nucleic acid of  claim 2 , wherein said monocotyledon plant is selected from a group consisting of maize, rice, wheat, and barley.  
     
     
         15 . The enzymatic nucleic acid of  claim 3 , wherein said dicotyledon plant is selected from a group consisting of canola, sunflower, safflower, soybean, cotton, peanut, olive, sesame, cuphea, flax, jojoba, and grape.  
     
     
         16 . The enzymatic nucleic acid of  claim 1 , wherein said gene is involved in fatty acid biosynthesis in said plant.  
     
     
         17 . The enzymatic nucleic acid of  claim 16 , wherein said gene is Δ-9 desaturase.  
     
     
         18 . The enzymatic nucleic acid of any of claims  16  or  17 , wherein said plant is selected from a group consisting of maize, canola, flax, sunflower, cotton, peanuts, safflower, soybean and rice.  
     
     
         19 . The enzymatic nucleic acid of  claim 1 , wherein said gene is involved in starch biosynthesis in said plant.  
     
     
         20 . The enzymatic nucleic acid of  claim 19 , wherein said gene is granule bound starch synthase.  
     
     
         21 . The enzymatic nucleic acid of any of claims  19  or  20 , wherein said plant is selected from a group consisting of maize, potato, wheat, and cassava.  
     
     
         22 . The enzymatic nucleic acid of  claim 1 , wherein said gene is involved in caffeine synthesis.  
     
     
         23 . The enzymatic nucleic acid of  claim 22 , wherein said gene is selected from a group consisting of 7-methylguanosine and 3-methyl transferase.  
     
     
         24 . The enzymatic nucleic acid of any of claims  22  or  23 , wherein said plant is a coffee plant.  
     
     
         25 . The enzymatic nucleic acid of  claim 1 , wherein said gene is involved in nicotine production in said plant.  
     
     
         26 . The enzymatic nucleic acid of  claim 25 , wherein said gene is selected from a group consisting of N-methylputrescine oxidase and putrescine N-methyl transferase.  
     
     
         27 . The enzymatic nucleic acid of any of claims  25  or  26 , wherein said plant is a tobacco plant.  
     
     
         28 . The enzymatic nucleic acid of  claim 1 , wherein said gene is involved in fruit ripening process in said plant.  
     
     
         29 . The enzymatic nucleic acid of  claim 28 , wherein said gene is selected from a group consisting of ethylene-forming enzyme, pectin methyltransferase, pectin esterase, polygalacturonase, 1-aminocyclopropane carboxylic acid (ACC) synthase, and ACC oxidase.  
     
     
         30 . The enzymatic nucleic acid of any of claims  28  or  29 , wherein said plant is selected from a group consisting of apple, tomato, pear, plum and peach.  
     
     
         31 . The enzymatic nucleic acid of  claim 1 , wherein said gene is involved in flower pigmentation in said plant.  
     
     
         32 . The enzymatic nucleic acid of  claim 31 , wherein said gene is selected from a group consisting of chalcone synthase, chalcone flavanone isomerase, phenylalanine ammonia lyase, dehydroflavonol hydroxylases, and dehydroflavonol reductase.  
     
     
         33 . The enzymatic nucleic acid of any of claims  31  or  32 , wherein said plant is selected from a group consisting of rose, petunia, chrysanthemum, and marigold.  
     
     
         34 . The enzymatic nucleic acid of  claim 1 , wherein said gene is involved in lignin production in said plant.  
     
     
         35 . The enzymatic nucleic acid of  claim 34 , wherein said gene is selected from a group consisting of O-methyltransferase, cinnamoyl-CoA:NADPH reductase and cinnamoyl alcohol dehydrogenase.  
     
     
         36 . The enzymatic nucleic acid of any of claims  34  or  35 , wherein said plant is selected from a group consisting of tobacco, aspen, poplar, and pine.  
     
     
         37 . A nucleic acid fragment comprising a cDNA sequence coding for maize Δ-9 desaturase, wherein said sequence is represented by the sequence I.D. No. 1.  
     
     
         38 . The enzymatic nucleic acid molecule of  claim 17 , wherein said nucleic acid specifically cleaves any of sequences defined in Table VI, wherein said nucleic acid is in a hammerhead configuration.  
     
     
         39 . The enzymatic nucleic acid molecule of  claim 17 , wherein said nucleic acid specifically cleaves any of sequences defined in Table VIII, wherein said nucleic acid is in a hairpin configuration.  
     
     
         40 . The enzymatic nucleic acid molecule of any of claims  38  or  39 , consisting essentially of one or more sequences selected from the group shown in Tables VII and VIII.  
     
     
         41 . The enzymatic nucleic acid molecule of  claim 20 , wherein said nucleic acid specifically cleaves any of sequences defined in Table IIIA, wherein said nucleic acid is in a hammerhead configuration.  
     
     
         42 . The enzymatic nucleic acid molecule of  claim 20 , wherein said nucleic acid specifically cleaves any of sequences defined in Tables VA and VB, wherein said nucleic acid is in a hairpin configuration.  
     
     
         43 . The enzymatic nucleic acid molecule of any of claims  41  or  42 , consisting essentially of one or more sequences selected from the group shown in Tables IIIB, IV, VA and VB.  
     
     
         44 . The enzymatic nucleic acid molecule of  claim 41 , consisting essentially of sequences defined as any of SEQ. I.D. NOS. 2-24.  
     
     
         45 . A plant cell comprising the enzymatic nucleic acid molecule of any of claims  1 - 8 ,  11 - 17 ,  19 - 20 ,  22 - 23 ,  25 - 26 ,  28 - 29 ,  31 - 32 ,  34 - 35 ,  37 - 39 ,  41 - 42  or  44 .  
     
     
         46 . A transgenic plant and the progeny thereof, comprising the enzymatic nucleic acid molecule of any of claims  1 - 8 ,  11 - 17 ,  19 - 20 ,  22 - 23 ,  25 - 26 ,  28 - 29 ,  31 - 32 ,  34 - 35 ,  37 - 39 ,  41 - 42  or  44 .  
     
     
         47 . An expression vector comprising nucleic acid encoding the enzymatic nucleic acid molecule of any of claims  1 - 8 ,  11 - 17 ,  19 - 20 ,  22 - 23 ,  25 - 26 ,  28 - 29 ,  31 - 32 ,  34 - 35 ,  37 - 39 ,  41 - 42  or  44 , in a manner which allows expression and/or delivery of that enzymatic nucleic acid molecule within a plant cell.  
     
     
         48 . An expression vector comprising nucleic acid encoding a plurality of enzymatic nucleic acid molecules of any of claims  1 - 8 ,  11 - 17 ,  19 - 20 ,  22 - 23 ,  25 - 26 ,  28 - 29 ,  31 - 32 ,  34 - 35 ,  37 - 39 ,  41 - 42  or  44 , in a manner which allows expression and/or delivery of said enzymatic nucleic acid molecules within a plant cell.  
     
     
         49 . A plant cell comprising the expression vector of  claim 47 .  
     
     
         50 . A plant cell comprising the expression vector of  claim 48 .  
     
     
         51 . A transgenic plant and the progeny thereof, comprising the expression vector of  claim 47 .  
     
     
         52 . A transgenic plant and the progeny thereof, comprising the expression vector of  claim 48 .  
     
     
         53 . A plant cell comprising the enzymatic nucleic acid of any of claims  16  or  17 .  
     
     
         54 . The plant cell of  claim 53 , wherein said cell is a maize cell.  
     
     
         55 . The plant cell of  claim 53 , wherein said cell is a canola cell.  
     
     
         56 . A transgenic plant and the progeny thereof, comprising the enzymatic nucleic acid of any of claims  16  or  17 .  
     
     
         57 . The transgenic plant and the progeny thereof of  claim 56 , wherein said plant is a maize plant.  
     
     
         58 . The transgenic plant and the progeny thereof of  claim 56 , wherein said plant is a canola plant.  
     
     
         59 . A plant cell comprising the enzymatic nucleic acid of any of claims  19  or  20 .  
     
     
         60 . The plant cell of  claim 59 , wherein said cell is a maize cell.  
     
     
         61 . A transgenic plant and the progeny thereof, comprising the enzymatic nucleic acid of any of claims  19  or  20 .  
     
     
         62 . The transgenic plant and progeny thereof of  claim 61 , wherein said plant is a maize plant.  
     
     
         63 . A method for modulating expression of an gene in a plant by administering to said plant the enzymatic nucleic acid molecule of any of claims  1 - 8 .  
     
     
         64 . The method of  claim 63 , wherein said plant is a monocot plant.  
     
     
         65 . The method of  claim 63 , wherein said plant is a dicot plant.  
     
     
         66 . The method of  claim 63 , wherein said plant is a gymnosperm.  
     
     
         67 . The method of  claim 63 , wherein said plant is an angiosperm.  
     
     
         68 . The method of  claim 63 , wherein said gene is Δ-9 desaturase.  
     
     
         69 . The method of  claim 68 , wherein said plant is a maize plant.  
     
     
         70 . The method of  claim 68 , wherein said plant is a canola plant.  
     
     
         71 . The method of  claim 63 , wherein said gene is granule bound starch synthase.  
     
     
         72 . The method of  claim 71 , wherein said plant is a maize plant.  
     
     
         73 . The expression vector of  claim 47 , wherein said vector comprises: 
 a) a transcription initiation region,    b) a transcription termination region;    c) a gene encoding at least one said enzymatic nucleic acid molecule; and,    wherein said gene is operably linked to said initiation region and said termination region, in a manner which allows expression and/or delivery of said enzymatic molecule within said plant cell.    
     
     
         74 . The expression vector of  claim 47 , wherein said vector comprises: 
 a) a transcription initiation region;    b) a transcription termination region;    c) an open reading frame;    d) a gene encoding at least one said enzymatic nucleic acid molecule, wherein said gene is operably linked to the 3′-end of said open reading frame; and,    wherein said gene is operably linked to said initiation region, said open reading frame and said termination region, in a manner which allows expression and/or delivery of said enzymatic molecule within said plant cell.    
     
     
         75 . The expression vector of  claim 47 , wherein said vector comprises: 
 a) a transcription initiation region;    b) a transcription termination region;    c) an intron;    d) a gene encoding at least one said enzymatic nucleic acid molecule; and,    wherein said gene is operably linked to said initiation region, said intron and said termination region, in a manner which allows expression and/or delivery of said enzymatic molecule within said plant cell.    
     
     
         76 . The expression vector of  claim 47 , wherein said vector comprises: 
 a) a transcription initiation region;    b) a transcription termination region;    c) an intron;    d) an open reading frame;    e) a gene encoding at least one said enzymatic nucleic acid molecule, wherein said gene is operably linked to the 3′-end of said open reading frame; and, wherein said gene is operably linked to said initiation region, said intron, said open reading frame and said termination region, in a manner which allows expression and/or delivery of said enzymatic molecule within said plant cell.    
     
     
         77 . The enzymatic nucleic acid of  claim 1 , wherein said plant is selected from the group consisting of maize, rice, soybeans, canola, alfalfa, cotton, wheat, barley, sunflower, flax and peanuts.  
     
     
         78 . A transgenic plant comprising nucleic acids encoding for an enzymatic nucleic acid molecule with RNA cleaving activity, wherein said nucleic acid molecule modulates the expression of a gene in said plant.  
     
     
         79 . The transgenic plant of  claim 78 , wherein said Plant is selected from the group consisting of maize, rice, soybeans, canola, alfalfa, cotton, wheat, barley, sunflower, flax and peanuts.  
     
     
         80 . The transgenic plant of  claim 78 , wherein said gene is granule bound starch synthase (GBSS).  
     
     
         81 . The transgenic plant of  claim 78 , wherein said gene is delta 9 desaturase.  
     
     
         82 . The transgenic plant of  claim 78 , wherein the plant is transformed with Agrobacteriurn, bombarding with DNA coated microprojectiles, whiskers, or electroporation.  
     
     
         83 . The transgenic plant of  claim 82 , wherein said bombarding with DNA coated microprojectiles is done with the gene gun.  
     
     
         84 . The transgenic plant of any of claims  78  or  82 , wherein said plant contains a selectable marker selected from the group consisting of chlorosulfuron, hygromycin, bar gene, bromoxynil, and kanamycin and the like.  
     
     
         85 . The transgenic plant of any of claims  78  or  82 , wherein said nucleic acid is operably linked to a promoter selected from the group consisting of octopine synthetase, the nopaline synthase, the manopine synthetase, cauliflower mosaic virus (35S); ribulose-1, 6-biphosphate (RUBP) carboxylase small subunit (ssu), the beta-conglycinin, the phaseolin promoter, napin, gamma zein, globulin, the ADH promoter, heat-shock, actin, and ubiquitin.  
     
     
         86 . The transgenic plant of  claim 78 , said enzymatic nucleic acid molecule is in a hammerhead, hairpin, hepatitis Δ virus, group I intron, group II intron, VS nucleic acid or RNaseP nucleic acid configuration  
     
     
         87 . The transgenic plant of  claim 86 , wherein said enzymatic nucleic acid with RNA cleaving activity encoded as a monomer.  
     
     
         88 . The transgenic plant of  claim 86 , wherein said enzymatic nucleic acid with RNA cleaving activity encoded as a multimer.  
     
     
         89 . The transgenic plant of  claim 78 , wherein the nucleic acids encoding for said enzymatic nucleic acid molecule with RNA cleaving activity is operably linked to the 3′ end of an open reading frame.  
     
     
         90 . The transgenic plant of  claim 78 , wherein said gene is an endogenous gene.  
     
     
         91 . A transgenic maize plant comprising in the 5′ to 3′ direction of transcription: 
 a promoter functional in said plant;  
 a double strand DNA (dsDNA) sequence encoding for a delta 9 gene of SEQ ID. No. 1, wherein transcribed strand of said dsDNA is complementary to RNA endogenous to said plant; and  
 a termination region functional in said plant.  
 
     
     
         92 . A transgenic maize plant comprising in the 5′ to 3′ direction of transcription, 
 a promoter functional in said plant;  
 a double strand DNA (dsDNA) sequence encoding for a granule bound starch synthase (GBSS) gene of SEQ ID NO. 25, wherein transcribed strand of said dsDNA is complementary to RNA endogenous to said plant; and  
 a termination region functional in said plant.  
 
     
     
         93 . The enzymatic nucleic acid molecule of  claim 1 , wherein said gene is an endogenous gene.  
     
     
         94 . The method of modulating expression of a gene of  claim 63 , wherein siad gene is an endogenous gene.  
     
     
         95 . The vector of FIG. 42, wherein said vector is employed for transformation of a plant cell.