Manipulation of cellulose and/or beta-1,4-glucan

Abstract

The present invention relates generally to isolated genes which encode polypeptides involved in cellulose biosynthesis in plants and transgenic plants expressing same in sense or antisense orientation, or as ribozymes, co-suppression or gene-targeting molecules. More particularly, the present invention is directed to a nucleic acid molecule isolated from Arabidopsis thaliana, Oryza sativa , wheat, barley, maize, Brassica spp., Gossypium hirsutum and Eucalyptus spp. which encode or an enzyme which is important in cellulose biosynthesis, in particular the cellulose synthase enzyme and homologues, analogues and derivatives thereof and uses of same in the production of transgenic plants expressing altered cellulose biosynthetic properties.

1 . An isolated nucleic acid molecule derived from an organism other than  Agrobacterium tumefaciens, Acetobacter pasteurianus  or  Acetobacter xylinum  which encodes a polypeptide of the cellulose biosynthetic pathway of a complementary sequence thereto, wherein said polypeptide is capable of producing cellulose and/or β-1,4-glucan polymer and/or an intermediate between cellulose and or β-glucan polymer.  
     
     
         2 . The isolated nucleic acid molecule according to  claim 1  wherein the polypeptide is cellulose synthase or a catalytic subunit thereof.  
     
     
         3 . The isolated nucleic acid molecule according to  claim 1 , derived from a plant.  
     
     
         4 . The isolated nucleic acid molecule according to  claim 3 , wherein the plant is selected from the list comprising  Arabidopsis thaliana, Gossypium hirsutum  (cotton),  Oryza sativa  (rice), wheat, barley, maize,  Brassica  ssp.,  Eucalyptus  ssp., hemp, jute, flax,  Pinus  ssp.,  Populus  ssp., and  Picea  spp.  
     
     
         5 . The isolated nucleic acid molecule according to  claim 2  wherein the cellulose synthase or catalytic subunit thereof is the  Arabidopsis thaliana  RSW1 polypeptide.  
     
     
         6 . The isolated nucleic acid molecule according to  claim 1 , comprising a sequence of nucleotides which is at least 40% identical to any one of SEQ ID NOs:1, 3, 4, 5, 7, 9, 11 or 13 or a complementary sequence thereof.  
     
     
         7 . An isolated nucleic acid molecule which encodes a cellulose synthase polypeptide or a catalytic subunit thereof and which: 
 (i) comprises the sequence of nucleotides set forth in any one of SEQ ID NOs:3, 4, 5, 7, 9 or 11;    (ii) hybridizes to 20 continuous nucleotides of any one of SEQ ID NOs:1, 3, 4, 5, 7, 9, 11 or 13 or a complementary sequence thereto under any of the following hybridization conditions: 
 (i) 6×SSC buffer, 0.1% (w/v) SDS at 28° C.;  
 (ii) 0.2×SSC-2×SSC buffer, 0.1% (w/v) SDS at 42° C. to 65° C.;  
 (iii) 0.1×SSC-0.2×SSC buffer, 0.1% (w/v) SDS at 55° C. to 65° C.; or  
   (iii) is amplifiable in a polymerase chain reaction using two primers that comprise 20-30 contiguous nucleotides derived from any one of SEQ ID NOs:1, 3, 4, 5, 7, 9, 11 or 13 or a complementary sequence thereto,    wherein said isolated nucleic acid molecule is derived from an organism other than  Agrobacterium trumefaciens, Acetobacter pasteurianus  or  Acetobacter xylinum.      
     
     
         8 . The isolated nucleic acid molecule according to  claim 7 , derived from a plant.  
     
     
         9 . The isolated nucleic acid molecule according to  claim 8 , wherein the plant is selected from the list comprising  Arabidopsis thaliana, Gossypium hirsutum  (cotton),  Oryza sativa  (rice), wheat, barley, maize,  Brassica  ssp.,  Eucalyptus  ssp., hemp, jute, flax,  Pinus  ssp.,  Populus  ssp., and  Picea  spp.  
     
     
         10 . The isolated acid molecule according to  claim 7 , wherein the cellulose synthase or catalytic subunit thereof is the  Arabidopsis thaliana  RSW1 polypeptide.  
     
     
         11 . The isolated nucleic acid molecule according to  claim 7 , comprising the protein-encoding region of any one of SEQ ID NOs:1, 3, 4, 5, 7, 9, 11 or 13.  
     
     
         12 . An isolated nucleic acid molecule derived from an organism other than  Agrobacterium tumefaciens, Acetobacter pasteurianus  or  Acetobacter xylinum  and which encodes a polypeptide of the cellulose biosynthetic pathway that is capable of producing cellulose and/or β-1,4-glucan and/or an intermediate between cellulose and a β-1,4-glucan polymer wherein said polypeptide comprises a sequence of amino acids which is at least 40% identical to any one of SEQ ID NOs:2, 6, 8, 10 12 or 14.  
     
     
         13 . A genetic construct which comprises the isolated nucleic acid molecule according to  claim 1 .  
     
     
         14 . A genetic construct which comprises the isolated nucleic acid molecule according to  claim 7 .  
     
     
         15 . The genetic construct according to  claim 14 , wherein the nucleic acid molecule is operably connected to a promoter sequence in the sense orientation such that RNA which encodes a cellulose synthase polypeptide or a catalytic subunit thereof is capable of being produced when said nucleic acid molecule is expressed in a cell containing said genetic construct.  
     
     
         16 . The genetic construct according to  claim 14 , wherein the nucleic acid molecule is operably connected to a promoter sequence in the antisense orientation such that RNA which is complementary to RNA which encodes a cellulose synthase polypeptide or a catalytic subunit thereof, is capable of being produced when said nucleic acid molecule is expressed in a cell containing said genetic construct.  
     
     
         17 . The genetic construct according to  claim 15 , wherein the promoter is the CaMV 35S promoter or the  Arabidopsis thaliana  RSW1 gene promoter.  
     
     
         18 . A method of increasing the level of cellulose in a cell, tissue, organ or organism, said method comprising expressing the isolated nucleic acid molecule according to  claim 1  therein, in the sense orientation, for a time and under conditions sufficient to produce or increase expression of the polypeptide encoded therefor.  
     
     
         19 . A method of reducing the level of non-crystaline β-1,4-glucan in a cell, tissue, organ or organism, said method comprising expressing the isolated nucleic acid molecule according to  claim 1  therein, in the sense orientation, for a time and under conditions at least sufficient to produce or increase expression of the polypeptide encoded therefor.  
     
     
         20 . A method of reducing the level of starch in a cell, tissue, organ or organism, said method comprising expressing the isolated nucleic acid molecule according to  claim 1  therein, in the sense orientation, for a time and under conditions at least sufficient to produce or increase expression of the polypeptide encoded therefor.  
     
     
         21 . A method of reducing the level of cellulose in a cell, tissue, organ or organism, said method comprising expressing the isolated nucleic acid molecule according to  claim 1  therein, in the antisense orientation, for a time and under conditions at least sufficient to prevent or reduce the expression of the polypeptide encoded thereof.  
     
     
         22 . A method of increasing the level of non-crystalline β-1,4-glucan in a cell, tissue, organ or organism, said method comprising expressing the isolated nucleic acid molecule according to  claim 1  therein, in the antisense orientation, for a time and under conditions at least sufficient to prevent or reduce the expression of the polypeptide encoded therefor.  
     
     
         23 . A method of increasing the level of starch in a cell, tissue, organ or organism, said method comprising expressing the isolated nucleic acid molecule according to  claim 1  therein, in the antisense orientation, for a time and under conditions at least sufficient to prevent or reduce the expression of the polypeptide encoded therefor.  
     
     
         24 . A method of producing a recombinant enzymatically active polypeptide which is capable of synthesizing cellulose and/or β-1,4-glucan and/or an intermediate between cellulose and α-1,4-glucan in a cell, said method comprising expressing the isolated nucleic acid molecule according to  claim 1  or a homologue, analogue or derivative thereof in said cell for a time and under conditions sufficient for the polypeptide encoded therefor to be produced.  
     
     
         25 . A recombinant polypeptide produced according to the method defined by  claim 24 .  
     
     
         26 . A recombinant cellulose biosynthetic polypeptide in substantially homogeneous form and capable of cellulose and/or β-1,4-glucan production and comprising a sequence of amino acids set forth in any one of SEQ ID NOs:2, 6, 8, 10, 12 or 14 or a homologue, analogue or derivative thereof which is at least 40% identical thereto.  
     
     
         27 . A method of altering the mechanical properties of a cell wall, said method comprising expressing the isolated nucleic acid molecule according to  claim 1  in the antisense orientation in said cell for a time and under conditions sufficient for the level of non-crystalline β-1,4-glucan to increase in said cell.  
     
     
         28 . An antibody molecule which binds to the recombinant polypeptide according to  claim 25 .  
     
     
         29 . A transgenic plant transformed with the isolated nucleic acid molecule according to  claim 1  or a genetic construct comprising said nucleic acid molecule.