Novel plastid-targeting nucleic acid sequence, a novel beta-amylase sequence, a stimulus-responsive promoter and uses thereof

Abstract

The invention provides a novel chloroplast targeted novel β-amylase sequence (ct β-amylase), a novel chloroplast targeting nucleic acid sequence and a novel β-amylase sequence. There is also disclosed an inducible promoter which is independently stimulated by light or sugar stimulus. Methods of transforming plants using these sequences are described, as well as transformed plant cells, transformed plants and seed thereof, as well as chimaeric genes containing the sequences. Modification of starch levels in plants can be achieved, as well as the targeting of genes from the starch biosynthetic or degradative pathways, disease or pest resistance or variation of gene expression due to stimulus are described.

1 . A nucleic acid sequence known herein as SEQ. ID. No. 1 and being from 1-294 nucleotides and having therewithin a sequence capable of targeting a further coding sequence to a plant plastid, or sequences being at least 65% or more homologous with the disclosed sequence SEQ. ID. No. 1 and having the same targeting ability.  
     
     
         2 . A nucleic acid sequence according to  claim 1 , wherein said nucleic acid sequence encodes about 94 amino acid residues.  
     
     
         3 . A nucleic acid sequence according to  claim 2 , wherein said sequence encodes about 85 amino acid residues.  
     
     
         4 . A nucleic acid sequence known herein as SEQ. ID. No. 2 and being from 1-1642 nucleotides and having therewithin a sequence capable of encoding β-amylase, or sequences being at least 65% or more homologous with the disclosed sequence within SEQ. ID. No. 2 and having the same encoding ability.  
     
     
         5 . A nucleic acid sequence known herein as SEQ. ID. No. 3 and being from 1-1953 nucleotides and having therewithin a sequence capable of encoding chloroplast targeted β-amylase, or sequences being at least 65% or more homologous with the disclosed sequence within SEQ. ID. No. 3 and having the same encoding ability.  
     
     
         6 . A nucleic acid sequence capable of directing expression of a product encoded by a coding sequence which is operably linked thereto, said nucleic acid sequence being known herein as SEQ. ID. No. 8, or being at least 65% homologous therewith and having substantially the same function thereas, and said nucleic acid sequence being responsive to stimulus, the level of expression of said product being variable in response to the stimulus applied to said nucleic acid sequence.  
     
     
         7 . A nucleic acid sequence according to any one of the preceding claims, wherein said nucleic acid sequence is an mRNA, cDNA or a genomic DNA sequence.  
     
     
         8 . A method of increasing or decreasing in a plant the activity of an enzyme in the pathway of starch biosynthesis or degradation, the method comprising the steps of stably incorporating into a plant genome a chimaeric gene comprising a nucleic acid sequence encoding a plastid targeting sequence and a coding sequence for an enzyme in the starch biosynthetic or degradative pathway, and regenerating a plant having an altered genome.  
     
     
         9 . A method according to  claim 8 , wherein said nucleic acid sequence comprises the sequence of  claim 1 .  
     
     
         10 . A method according to  claim 8 , wherein said enzyme in the starch degradative pathway is β-amylase.  
     
     
         11 . A method according to  claim 8 ,  9  or  10 , wherein said coding sequence is the nucleic acid sequence of  claim 4 , being a coding sequence for an enzyme in the starch degradation pathway.  
     
     
         12 . A method according to  claim 8 , wherein said nucleic acid sequence is the nucleic acid sequence of  claim 5 .  
     
     
         13 . A method according to any one of claims  8 - 12 , wherein said enzyme from the starch biosynthetic pathway is one of the group consisting of sucrose synthase, ADPG pyrophosphorylase, starch synthase, branching enzyme, α amylase, isoamylase, non-plastidic β-amylase, α-glucosidase, starch phosphorylase and disproportionating enzyme.  
     
     
         14 . A method of targeting proteins or enzymes to a plant plastid, the method comprising the steps of stably incorporating into a plant genome a chimaeric gene comprising a nucleic acid sequence encoding a plastid targeting sequence and a coding sequence for a protein or an enzyme, and regenerating a plant having an altered genome, the protein or enzyme being one or more in the pathway of the following group: lipid synthesis, photosynthesis, amino acid metabolism, nitrogen fixation, carbon fixation or synthesis of carbohydrate polymers; or being able to confer a characteristic to the plant, the nucleic acid sequence being the nucleic acid sequence of  claim 1 .  
     
     
         15 . A method according to  claim 14 , wherein said characteristics selected from one or more of the following group: herbicide resistance and pest resistance.  
     
     
         16 . A method of varying the level of expression of a product encoded by a coding sequence operably linked to a nucleic acid sequence capable of directing expression of said product in a plant, said method comprising the steps of stably incorporating into a plant genome a chimaeric gene comprising a nucleic acid sequence capable of directing expression of a product encoded by a coding sequence that is operably linked thereto, said nucleic acid sequence having substantially the sequence of SEQ. ID. No. 8 or being at least 65% homologous therewith and having substantially the same function thereas, and being responsive to stimulus.  
     
     
         17 . A method according to  claim 16 , wherein said coding sequence encodes a gene causing sterility in said plant during flowering and which is caused to be expressed by said nucleic acid sequence upon exposure to light.  
     
     
         18 . A method according to  claim 16 , wherein said coding sequence encodes a gene which alters the level of starch biosynthetic enzyme genes in leaves or other photosynthetic tissue.  
     
     
         19 . A method according to  claim 16 , wherein said coding sequence encodes a gene whose expression level is varied upon the generation of sugar in a developing storage organ and the effect of sugar on said nucleic acid sequence.  
     
     
         20 . A method according to  claim 16 , wherein said nucleic acid sequence is operable when there is light but no sugar.  
     
     
         21 . A method according to  claim 16 , wherein said nucleic acid sequence is operable when there is sugar but no light.  
     
     
         22 . A method of altering in a transgenic plant, which plant already shows an increase or decrease in an enzyme activity as a result of genetic transformation, a further enzyme in order to up or down regulate said further enzyme and thereby increase or decrease the amount of starch produced by the retransformed plant.  
     
     
         23 . A method according to  claim 22 , wherein said first transformed plant is a plant having an increased or decreased enzyme activity in the starch biosynthetic pathway.  
     
     
         24 . A method according to  claim 23 , wherein said first transformed plant is a transgenic potato transformed with the gene for ADPG-PPase.  
     
     
         25 . A method according to any one of claims  8 ,  14  or  16 , wherein said chimaeric gene further comprises a promoter selected from the group consisting of the cauliflower mosaic virus 35S promoter (full or truncated), the rubisco promoter, the pea plastocyanin promoter, the nopaline synthase promoter, the chlorophyll r/b binding promoter, the high molecular weight glutenin promoter, the α, β-gliadin promoter, the hordein promoter and the patatin promoter.  
     
     
         26 . A method according to any one of claims  8 ,  14  or  16 , wherein said coding sequence of said enzyme in said chimaeric gene provides up or down regulation of the activity of said enzyme.  
     
     
         27 . A method according to  claim 16 , wherein said stimulus is the presence or absence of light and/or varying levels of sugar.  
     
     
         28 . A method according to  claim 16 , wherein said stimulus is a stimulus which is developmentally controlled.  
     
     
         29 . A method according to  claim 27 , wherein the sugar is one or more of sucrose or glucose.  
     
     
         30 . A method according to  claim 29 , wherein the sugar is sucrose.  
     
     
         31 . A chimaeric gene comprising a nucleic acid sequence according to  claim 1 .  
     
     
         32 . A chimaeric gene comprising a nucleic acid sequence encoding a plastid targeting sequence according to  claim 1  and a nucleic acid coding sequence of an enzyme in the starch, degradative pathway, the enzyme being encoded by the sequence according to  claim 4 .  
     
     
         33 . A chimaeric gene comprising a nucleic acid sequence capable of directing expression of a further coding sequence, said coding sequence being the sequence according to  claim 4 .  
     
     
         34 . A chimaeric gene comprising a nucleic acid sequence according to  claim 6  that is responsive to stimulus and a coding sequence, the level of expression of said coding sequence being variable in response to the stimulus applied to said nucleic acid sequence.  
     
     
         35 . Plant cells comprising the nucleic acid sequence of any one of claims  1 ,  4 ,  5  or  6 .  
     
     
         36 . Seed of the transformed plant containing one or more of the nucleic acid sequences of claims  1 ,  4 ,  5 , or  6 .  
     
     
         37 . Plants transformed according to the methods of any one of claims  8 ,  14  or  16 , where in said plants are one or more of the group consisting of potato, wheat, maize, barley, tomato, rice, pea, soybean, peanut, cassava, yam, banana and tobacco.