Lipase polypeptide

Abstract

We describe a plant lipase polypeptide and nucleic acids that encode said polypeptide which has homology to a patatin and which has phospholipase and/or triacylglycerol lipase activity.

1 . An isolated cell wherein said cell is a transgenic cell and is transformed with a vector comprising a nucleic acid molecule wherein said nucleic acid molecule is selected from the group consisting of:
 i) a nucleic acid molecule comprising a nucleic acid sequence as represented in  FIG. 1   a,    9   a  or  10   a;      ii) a nucleic acid molecule which hybridises under stringent hybridisation conditions to the nucleic acid molecule(s) in (i) and which encodes a polypeptide that has phospholipase and/or triacylglycerol lipase activity;   iii) a nucleic acid molecule that encodes a polypeptide comprising an amino acid sequence as represented in  FIG. 1   b,    9   b  or  10   b,  or a variant polypeptide which is modified by addition, deletion or substitution of at least one amino acid residue wherein said polypeptide has phospholipase and/or triacylglycerol lipase activity.   
     
     
         2 . A transgenic cell according to  claim 1  wherein said cell is transformed with a vector wherein said vector includes a nucleic acid molecule consisting of the nucleic acid sequence represented in  FIG. 1   a,    9   a  or  10   a.    
     
     
         3 . A transgenic cell according to  claim 1  or  2  wherein said vector is an expression vector adapted for expression of said nucleic acid molecule. 
     
     
         4 . A transgenic cell according to  claim 3  wherein said adaptation is the provision of a cell or tissue specific promoter. 
     
     
         5 . A transgenic cell according to  claim 4  wherein said promoter is an inducible promoter or a developmentally regulated promoter. 
     
     
         6 . A transgenic cell according to any of  claims 1 - 5  wherein said cell is eukaryotic cell. 
     
     
         7 . A transgenic cell according to  claim 6  wherein said eukaryotic cell is a plant cell. 
     
     
         8 . A transgenic cell according to any of  claims 1 - 7  wherein said cell is transformed with at least one further nucleic acid molecule wherein said nucleic acid molecule is selected from the following group:
 i) a nucleic acid molecule comprising a nucleic acid sequence as represented in  FIG. 3  or  4 ; 
 ii) a nucleic acid molecule which hybridises to the nucleic acid molecule in (i) under stringent hybridisation conditions and which encodes a polypeptide that has triacylglycerol lipase activity; 
 iii) a nucleic acid molecule which differs from the nucleic acid molecules of (i) and (ii) due to the degeneracy in the genetic code. 
 
     
     
         9 . A transgenic cell according to  claim 8  wherein said nucleic acid molecule consists of the nucleic acid sequence represented in  FIG. 3  or  4 . 
     
     
         10 . A transgenic cell according to any of  claims 1 - 9  wherein said cell over-expresses the nucleic acid molecule(s) encoding said phospholipase and/or triacylglycerol lipase when compared to a non-transgenic reference cell of the same species. 
     
     
         11 . A transgenic cell according to any of  claims 1 - 9  wherein said cell is modified such that the expression of the nucleic acid molecule(s) encoding said phospholipase and/or triacylglycerol lipase activity is decreased when compared to a non-transgenic reference cell of the same species. 
     
     
         12 . A transgenic cell according to  claim 11  wherein said transgenic cell is null for a nucleic acid molecule comprising a sequence selected from the group consisting of:
 i) a nucleic acid molecule comprising a sequence as represented by  FIGS. 1   a,    9   a,    10   a  and/or,  FIGS. 3  and/or  FIG. 4 ; 
 ii) a nucleic acid molecule that hybridise to the sequences of (i) above and which encodes a polypeptide that has phospholipase and/or triacylglycerol lipase activity; and 
 iii) nucleic acid sequences which are degenerate as a result of the genetic code to the sequences defined in (i) and (ii) above. 
 
     
     
         13 . A plant cell wherein said cell is transformed with a nucleic acid molecule comprising an expression cassette which cassette comprises a nucleic acid sequence selected from the group consisting of:
 i) a nucleic acid molecule comprising a nucleic acid sequence as represented in  FIG. 1   a,    9   a  or  10   a;      ii) a nucleic acid molecule which hybridises under stringent hybridisation conditions to the nucleic acid molecule(s) in (i) and which encodes a polypeptide that has phospholipase and/or triacylglycerol lipase activity;   iii) a nucleic acid molecule that encodes a polypeptide comprising an amino acid sequence as represented in  FIG. 1   b,    9   b  or  10   b,  or a variant polypeptide which is modified by addition, deletion or substitution of at least one amino acid residue wherein said polypeptide has phospholipase and/or triacylglycerol lipase activity; wherein said cassette is adapted such that both sense and antisense nucleic acid molecules are transcribed from said cassette.   
     
     
         14 . A plant cell according to  claim 13  wherein said cassette is provided with at least two promoters adapted to transcribe sense and antisense strands of said nucleic acid molecule. 
     
     
         15 . A plant cell according to  claim 13  or  14  wherein said cassette comprises a nucleic acid molecule wherein said molecule comprises a first part linked to a second part wherein said first and second parts are complementary over at least part of their sequence and further wherein transcription of said nucleic acid molecule produces an RNA molecule which forms a double stranded region by complementary base pairing of said first and second parts. 
     
     
         16 . A transgenic plant comprising a cell according to any of  claims 1 - 15 . 
     
     
         17 . A transgenic plant according to  claim 16  wherein said plant is an oil seed plant. 
     
     
         18 . A seed comprising a plant cell according to any of  claims 7 - 17 . 
     
     
         19 . A seed according to  claim 18  wherein said seed is from an oil seed plant. 
     
     
         20 . A cell according to any of  claims 1 - 10  wherein said cell is a prokaryotic cell. 
     
     
         21 . A method to manufacture a phospholipase and/or tracylglycerol lipase polypeptide comprising the steps of:
 i) providing a cell according to any of  claims 1 - 10  and growth conditions conducive to the production of said polypeptide; and optionally   ii) purifying said polypeptide from said cell or growth media.   
     
     
         22 . The use of a polypeptide encoded by a nucleic acid molecule selected from the group consisting of:
 i) a nucleic acid molecule comprising a nucleic acid sequence as represented in  FIG. 1   a,    9   a  or  10   a;      ii) a nucleic acid molecule which hybridises under stringent hybridisation conditions to the nucleic acid molecule(s) in (i) and which encodes a polypeptide that has phospholipase and/or triacylglycerol lipase activity;   iii) a nucleic acid molecule that encodes a polypeptide comprising an amino acid sequence as represented in  FIG. 1   b,    9   b  or  10   b,  or a variant polypeptide which is modified by addition, deletion or substitution of at least one amino acid residue wherein said polypeptide has phospholipase and/or triacylglycerol lipase activity, as a target for the discovery of agents that inhibit the lipase activity of said polypeptide.   
     
     
         23 . A screening method for the identification of an agent with the ability to inhibit plant growth and/or viability comprising the steps of: 
       i) providing a polypeptide encoded by the nucleic acid selected from the following group;
 a) a nucleic acid molecule comprising a nucleic acid sequence as represented in  FIG. 1   a,    9   a  or  10   a;    
 d) a nucleic acid molecule which hybridises to the nucleic acid molecule(s) in (i) and which encodes a polypeptide that has phospholipase and/or triacylglycerol lipase activity; 
 e) a nucleic acid molecule that encodes a polypeptide comprising an amino acid sequence as represented in  FIG. 1   b,    9   b  or  10   b,  or a variant polypeptide which is modified by addition, deletion or substitution of at least one amino acid residue wherein said polypeptide has phospholipase and/or triacylglycerol lipase activity; 
 
       ii) providing at least one candidate agent; 
       iii) forming a preparation which is a combination of (i) and (ii); 
       iv) determining the interaction of the polypeptide and said candidate agent; and 
       testing the effect of the agent on the growth and/or viability of plants. 
     
     
         24 . A method according to  claim 23  wherein said agent is a herbicide. 
     
     
         25 . A reaction vessel comprising the polypeptide encoded by a nucleic acid molecule selected from the group consisting of:
 i) a nucleic acid molecule comprising a nucleic acid sequence as represented in  FIG. 1   a,    9   a  or  10   a;      ii) a nucleic acid molecule which hybridises under stringent hybridisation conditions to the nucleic acid molecule(s) in (i) and which encodes a polypeptide that has phospholipase and/or triacylglycerol lipase activity;   iii) a nucleic acid molecule that encodes a polypeptide comprising an amino acid sequence as represented in  FIG. 1   b,    9   b  or  10   b,  or a variant polypeptide which is modified by addition, deletion or substitution of at least one amino acid residue wherein said polypeptide has phospholipase and/or triacylglycerol lipase activity, fatty acid substrates and co-factors.   
     
     
         26 . A vessel according to  claim 25  wherein said vessel comprises polypeptides comprising the amino acid sequences as represented by  FIGS. 1   a  or  9   a  and/or  10   a  and/or  FIG. 2  and/or  FIG. 3 , or sequence variants thereof. 
     
     
         27 . A vessel according to  claim 25  or  26  wherein said at least one polypeptide is expressed by a cell according to any of  claims 1 - 10 . 
     
     
         28 . A vessel according to  claim 25  or  26  wherein said polypeptide(s) is/are soluble. 
     
     
         29 . A vessel according to  claim 28  wherein said polypeptide(s) is/are immobilised. 
     
     
         30 . A vessel according to any of  claims 25 - 29  wherein said vessel is a bioreactor. 
     
     
         31 . A method to increase the fatty acid content of a seed comprising the steps of:
 i) cultivating a plant according to  claim 16  or  17  to produce seed;   ii) harvesting said seed from said plant; and optionally   iii) determining the fatty acid content of said harvested seed.   
     
     
         32 . An isolated cell wherein said cell is a transgenic cell and is transformed with a vector comprising a nucleic acid molecule wherein said nucleic acid molecule is selected from the group consisting of:
 i) a nucleic acid molecule comprising a nucleic acid sequence as represented in  FIG. 11   a;      ii) a nucleic acid molecule which hybridises under stringent hybridisation conditions to the nucleic acid molecule(s) in (i) and which has phospholipase and/or triacylglycerol lipase activity;   iii) a nucleic acid molecule that encodes a polypeptide comprising an amino acid sequence as represented in  FIG. 11   b,  or a variant polypeptide which is modified by addition, deletion or substitution of at least one amino acid residue wherein said polypeptide has phospholipase and/or triacylglycerol lipase activity.   
     
     
         33 . A plant cell wherein said cell is transformed with a nucleic acid molecule comprising an expression cassette which cassette comprises a nucleic acid sequence selected from the group consisting of:
 i) a nucleic acid molecule comprising a nucleic acid sequence as represented in  FIG. 11   a;      ii) a nucleic acid molecule which hybridises under stringent hybridisation conditions to the nucleic acid molecule(s) in (i) and which encodes a polypeptide that has phospholipase and/or triacylglycerol lipase activity;   iii) a nucleic acid molecule that encodes a polypeptide comprising an amino acid sequence as represented in  FIG. 11   b,  or a variant polypeptide which is modified by addition, deletion or substitution of at least one amino acid residue wherein said polypeptide has phospholipase and/or triacylglycerol lipase activity; wherein said cassette is adapted such that both sense and antisense nucleic acid molecules are transcribed from said cassette.   
     
     
         34 . A plant or seed comprising a plant cell according to  claim 33 . 
     
     
         35 . The use of a polypeptide encoded by a nucleic acid molecule selected from the group consisting of:
 i) a nucleic acid molecule comprising a nucleic acid sequence as represented in  FIG. 11   a;      ii) a nucleic acid molecule which hybridises under stringent hybridisation conditions to the nucleic acid molecule(s) in (i) and which encodes a polypeptide that has phospholipase and/or triacylglycerol lipase activity;   iii) a nucleic acid molecule that encodes a polypeptide comprising an amino acid sequence as represented in  FIG. 11   b,  or a variant polypeptide which is modified by addition, deletion or substitution of at least one amino acid residue wherein said polypeptide has phospholipase and/or triacylglycerol lipase activity, as a target for the discovery of agents that inhibit the lipase activity of said polypeptide.   
     
     
         36 . A screening method for the identification of an agent with the ability to inhibit plant growth and/or viability comprising the steps of: 
       i) providing a polypeptide encoded by the nucleic acid selected from the following group;
 a) a nucleic acid molecule comprising a nucleic acid sequence as represented in  FIG. 11   a;    
 f) a nucleic acid molecule which hybridises to the nucleic acid molecule(s) in (i) and which encodes a polypeptide that has phospholipase and/or triacylglycerol lipase activity; 
 g) a nucleic acid molecule that encodes a polypeptide comprising an amino acid sequence as represented in  FIG. 11   b,  or a variant polypeptide which is modified by addition, deletion or substitution of at least one amino acid residue wherein said polypeptide has phospholipase and/or triacylglycerol lipase activity; 
 
       ii) providing at least one candidate agent; 
       iii) forming a preparation which is a combination of (i) and (ii); 
       iv) determining the interaction of the polypeptide and said candidate agent; and 
       testing the effect of the agent on the growth and/or viability of plants. 
     
     
         37 . A reaction vessel comprising the polypeptide encoded by a nucleic acid molecule selected from the group consisting of:
 i) a nucleic acid molecule comprising a nucleic acid sequence as represented in  FIG. 11   a;      ii) a nucleic acid molecule which hybridises under stringent hybridisation conditions to the nucleic acid molecule(s) in (i) and which encodes a polypeptide that has phospholipase and/or triacylglycerol lipase activity;   iii) a nucleic acid molecule that encodes a polypeptide comprising an amino acid sequence as represented in  FIG. 11   b,  or a variant polypeptide which is modified by addition, deletion or substitution of at least one amino acid residue wherein said polypeptide has phospholipase and/or triacylglycerol lipase activity, fatty acid substrates and co-factors.   
     
     
         38 . A method to increase the fatty acid content of a seed comprising the steps of:
 i) cultivating a plant according to  claim 34  to produce seed;   ii) harvesting said seed from said plant; and optionally   iii) determining the fatty acid content of said harvested seed.