1-aminocyclopropane-1-carboxylate synthase gene from Rosa to control ethylene levels in roses

Abstract

A gene which encodes an ACC synthase is identified for the rose plants, specifically Rosa (cardinal red). This gene is shown as modified to achieve a transgenic plant which resists wilting and the like as a result of reduced ethylene production. This alteration is reproduced by the transformed plant. Isolation of high quality mRNA is achieved through use and adaptation of a 2-butoxyethanol precipitation technique using large amount of initial tissue in order to achieve critical mass for precipitation.

What is claimed is:  
     
         1 . An isolated DNA molecule consisting of SEQ ID NO: 1.  
     
     
         2 . An isolated DNA molecule encoding an ACC synthase enzyme of a rose which DNA molecule hybridizes under high stringency conditions with SEQ ID NO: 1.  
     
     
         3 . A vector useful when introduced into a rose plant cell, comprising an oligonucleotide or polynucleotide which is complementary to the nucleotide sequence SEQ ID NO: 1 or is complementary to an RNA sequence encoded by SEQ ID NO: 1.  
     
     
         4 . A vector useful when introduced into a rose plant cell, comprising an oligonucleotide or polynucleotide which is complementary to a nucleotide sequence which encodes an ACC synthase enzyme of a rose and which hybridizes under high stringency conditions with the nucleotide sequence SEQ ID NO: 1 or is complementary to an RNA sequence encoded by such nucleotide sequence.  
     
     
         5 . A vector according to  claim 3  or  4  and further comprising regulatory sequences required for expression of said oligonucleotide or polynucleotide in said cell.  
     
     
         6 . A vector according to  claim 5  wherein said regulatory sequences comprise a promoter active in said cell.  
     
     
         7 . A vector according to  claim 6 , wherein said regulatory sequences further comprise a polyadenylation signal.  
     
     
         8 . A vector according to  claim 6 , wherein said promoter comprises a heterologous promoter.  
     
     
         9 . A vector according to  claim 8 , wherein said heterologous promoter is a viral promoter.  
     
     
         10 . A vector according to  claim 9 , wherein said viral promoter is the CaMV 35S promoter or a promoter homologous to CaMV35S.  
     
     
         11 . A vector according to  claim 8 , wherein said heterologous promoter is selected from the group consisting of the SSU gene promoter, ribulose bisphosphate carboxylase promoter, chlorophyll a/b binding protein promoter, potato ST-LS1 gene promoter, soybean heat shock protein hsp17.5-E promoter, soybean heat shock protein hsp17.3-B promoter, phenylalanine ammonia-lyase promoter, petunia 5-enolpyruvylshikimate-3-phosphate synthase gene promoter,  Rhizobium meliloti  FIXD gene promoter and nopaline synthase promoter.  
     
     
         12 . A rose cell transformed with a vector according to  claim 3  or  4 .  
     
     
         13 . A mature rose plant regenerated from a cell transformed with a vector according to  claim 3  or  4 .  
     
     
         14 . A plant part of a rose plant according to  claim 13 .  
     
     
         15 . An isolated nucleic acid molecule encoding an amino acid sequence consisting of SEQ ID NO: 1.  
     
     
         16 . An isolated DNA molecule having substantial sequence homology with a molecule consisting of SEQ ID NO: 1.  
     
     
         17 . An isolated DNA molecule having substantial sequence homology with a molecule consisting of SEQ ID NO: 1 according to  claim 16  wherein said substantial sequence homology is selected from a group of percentages of similarity consisting of at least 70 percent, at least 80 percent, and at least 90 percent.  
     
     
         18 . An isolated DNA molecule according to  claim 16  selected from the group consisting of SEQ ID NO: 1.  
     
     
         19 . A method for genetically altering a plant, comprising the steps of: 
 (a) isolating mRNA of said plant using a 2-butoxyethanol precipitation technique with a critical mass amount of RNA for precipitation;    (b) constructing a cDNA library from said isolated mRNA;    (c) identifying and cloning a desired DNA sequence from said library;    (d) genetically altering said cloned DNA sequence; and    (e) transforming said plant with said altered DNA sequence, thereby genetically altering said plant.    
     
     
         20 . A method according to  claim 19 , wherein said step of isolating mRNA of said plant using a 2-butoxyethanol precipitation technique using a large amount of tissue from said plant comprises the step of using at least about 3 to 5 grams of tissue from said plant.  
     
     
         21 . A method for genetically altering a plant, comprising the steps of: 
 (a) isolating mRNA of said plant using a 2-butoxyethanol precipitation technique using a large amount of tissue from said plant;    (b) constructing a cDNA library from said isolated mRNA;    (c) identifying and cloning a desired DNA sequence from said library;    (d) genetically altering said cloned DNA sequence;    (e) transforming said plant with said altered DNA sequence, thereby genetically altering said plant.    
     
     
         22 . A method according to  claim 19 , wherein said plant is a member of a low RNA plant species.  
     
     
         23 . A method according to  claim 22 , wherein said plant is a species of the genus Pelargonium or Rosa.  
     
     
         24 . A method according to  claim 19 ,  20 , or  21  wherein said plant is selected from a group comprising woody plants.  
     
     
         25 . A method according to  claim 19 , wherein said cloned DNA sequence encodes ACC synthase.  
     
     
         26 . A method according to  claim 25 , wherein said plant is a rose plant.  
     
     
         27 . A method according to  claim 26 , wherein said cDNA is selected from the group consisting of SEQ ID NO: 1.  
     
     
         28 . A method according to  claim 19 , wherein said isolating step (a) comprises the steps of: 
 (a) contacting said RNA with a binding partner for mRNA; and    (b) obtaining said bound mRNA.    
     
     
         29 . A method for producing a genetically altered rose plant, comprising the steps of: 
 (a) isolating rose mRNA using a 2-butoxyethanol precipitation technique wherein at least about 3-5 grams of plant tissue starting material is used to attain a critical mass amount of RNA for precipitation;    (b) constructing a cDNA library from said isolated mRNA;    (c) identifying and cloning at least one DNA sequence from said library;    (d) genetically altering said cloned DNA sequence;    (e) transforming rose cells with said altered DNA sequence; and    (f) regenerating said genetically altered rose plant from said cells, which plant expresses said altered DNA sequence.    
     
     
         30 . A method of isolating plant mRNA, comprising the steps of: 
 (a) extracting nucleic acids from sufficient plant tissue to attain a critical mass amount of RNA for precipitation;    (b) isolating RNA from said nucleic acids of step (a) using a 2-butoxyethanol precipitation technique;    (c) contacting said RNA with a binding partner for mRNA; and    (d) obtaining said bound mRNA, thereby isolating said mRNA.    
     
     
         31 . A method of isolating plant mRNA according to claim  30  wherein said binding partner is immobilized on a solid carrier thereby generating immobilized mRNA and wherein said step of obtaining said bound mRNA comprises the step of eluting said mRNA from said carrier.  
     
     
         32 . A method of isolating plant mRNA according to claim  31  wherein said step of extracting nucleic acids from sufficient plant tissue to attain a critical mass amount of RNA for precipitation comprises the step of using at least about 3 to 5 grams of tissue from said plant.