Genes coding for tomato beta-galactosidase polypeptides

Abstract

A novel β-galactosidase gene family and DNA sequences derived from the cloning of cDNAs encoding products of these genes are provided, as exemplified by a β-galactosidase II protein which is encoded by a cDNA clone, pZBG2-1-4. A method for modifying cell wall metabolism which involves modifying the activity of at least one β-galactosidase, and thus modifying the quality of the fruit is also provided. Also provided by the present invention is a DNA construct including some or all of a β-galactosidase DNA sequence under control of a transcriptional initiation region operative in plants, so that the construct can generate RNA and, optionally, β-galactosidase polypeptide in plant cells. The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of β-galactosidase polypeptides or peptides by recombinant techniques. The present invention also provides plant cells containing DNA constructs of the present invention; plants derived therefrom having modified β-galactosidase gene expression; and seeds produced from such plants.

1 . An isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence at least 95% identical to a sequence selected from the group consisting of: 
 (a) a nucleotide sequence encoding the β-galactosidase II polypeptide having the complete amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO: 15 and SEQ ID NO: 16 and designated TBG1, TBG2, TBG3, TBG4, TBG5, TBG6 and TBG7, respectively as shown in  FIG. 2  or as encoded by the cDNA clone selected from the group consisting of cDNA clones contained in Gen Bank Accession No. AF023847, AF1544420, AF154421, AF020390, AF154423, AF154424 and AF154422;    (b) a nucleotide sequence encoding the mature β-galactosidase II polypeptide having the amino acid sequence at about positions 24-724 selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 and designated TBG1, TBG2, TBG3, TBG4, TBG5, TBG6 and TBG7, respectively as shown in  FIG. 2  or as encoded by the cDNA clone selected from the group consisting of cDNA clones contained in Gen Bank Accession No. AF023847, AF1544420, AF154421, AF020390, AF154423, AF154424 and AF154422; and    ( c) a nucleotide sequence complementary to any of the nucleotide sequences in (a) or (b), above.    
     
     
         2 . The nucleic acid molecule of  claim 1  wherein said polynucleotide has the complete nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7 as shown in  FIG. 2 .  
     
     
         3 . The nucleic acid molecule of  claim 1  wherein said polynucleotide has the nucleotide sequence in  FIG. 2  (SEQ ID NO:4) encoding the β-galactosidase II polypeptide having the amino acid sequence designated TBG4 in  FIG. 2 .  
     
     
         4 . The nucleic acid molecule of  claim 1  wherein said polynucleotide has the nucleotide sequence in  FIG. 2  (SEQ ID NO:4) encoding the mature polypeptide having the amino acid sequence from about 24 to about 724 in the amino acid sequence designated TBG4 in  FIG. 2 .  
     
     
         5 . The nucleic acid molecule of  claim 1  wherein said polynucleotide has the complete nucleotide sequence of the cDNA clone contained in Gen Bank Accession No. AF023847.  
     
     
         6 . The nucleic acid molecule of  claim 1  wherein said polynucleotide has the complete nucleotide sequence of the cDNA clone contained in Gen Bank Accession No. AF1544420.  
     
     
         7 . The nucleic acid molecule of  claim 1  wherein said polynucleotide has the complete nucleotide sequence of the cDNA clone contained in Gen Bank Accession No. AF154421.  
     
     
         8 . The nucleic acid molecule of  claim 1  wherein said polynucleotide has the complete nucleotide sequence of the cDNA clone contained in Gen Bank Accession No. AF020390.  
     
     
         9 . The nucleic acid molecule of  claim 1  wherein said polynucleotide has the complete nucleotide sequence of the cDNA clone contained in Gen Bank Accession No. AF154423.  
     
     
         10 . The nucleic acid molecule of  claim 1  wherein said polynucleotide has the complete nucleotide sequence of the cDNA clone contained in Gen Bank Accession No. AF154424.  
     
     
         11 . The nucleic acid molecule of  claim 1  wherein said polynucleotide has the complete nucleotide sequence of the cDNA clone contained in Gen Bank Accession No. AF154422.  
     
     
         12 . An isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide having a nucleotide sequence identical to a nucleotide sequence in (a), (b), or © of  claim 1  wherein said polynucleotide which hybridizes does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues.  
     
     
         13 . An isolated nucleic acid molecule comprising a polynucleotide which encodes the amino acid sequence of an epitope-bearing portion of a β-galactosidase II polypeptide having an amino acid sequence in (a), (b), or © of  claim 1 .  
     
     
         14 . A method for making a recombinant vector comprising inserting an isolated nucleic acid molecule of  claim 1  into a vector.  
     
     
         15 . A recombinant vector produced by the method of  claim 14 .  
     
     
         16 . A method of making a recombinant host cell comprising introducing the recombinant vector of  claim 15  into a host cell.  
     
     
         17 . A recombinant host cell produced by the method of  claim 16 .  
     
     
         18 . A recombinant method for producing β-galactosidase II polypeptide, comprising culturing the recombinant host cell of  claim 17  under conditions such that said polypeptide is expressed and recovering said polypeptide.  
     
     
         19 . An isolated β-galactosidase II polypeptide comprising an amino acid sequence at least 95% identical to a sequence selected from the group consisting of: 
 a) amino acid sequence at about positions 24-724 selected from the group consisting of sequences SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 and designated TBG1, TBG2, TBG3, TBG4, TBG5, TBG6 and TBG7, respectively as shown in  FIG. 2 ; and    b) amino acid sequence as encoded by the cDNA clone selected from the group consisting of cDNA clones contained in Gen Bank Accession No. AF023847, AF1544420, AF154421, AF020390, AF154423, AF154424 and AF154422.    
     
     
         20 . An isolated polypeptide comprising an epitope-bearing portion of the β-galactosidase II protein.  
     
     
         21 . An isolated antibody that binds specifically to a β-galactosidase II polypeptide of  claim 20 .  
     
     
         22 . An isolated nucleic acid molecule nucleic acid molecule comprising a polynucleotide having a nucleotide sequence at least 95% identical to a sequence selected from the group consisting of: 
 (a) a nucleotide sequence encoding the β-galactosidase II polypeptide having the complete amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO: 15 and SEQ ID NO: 16 and designated TBG1, TBG2, TBG3, TBG4, TBG5, TBG6 and TBG7, respectively as shown in  FIG. 3  or as encoded by the cDNA clone selected from the group consisting of cDNA clones contained in Gen Bank Accession No. AF023847, AF1544420, AF154421, AF020390, AF154423, AF154424 and AF154422;    (b) a nucleotide sequence encoding the mature β-galactosidase II polypeptide having the amino acid sequence at about positions 24-724 selected from the group consisting of sequences SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 and designated TBG1, TBG2, TBG3, TBG4, TBG5, TBG6 and TBG7, respectively as shown in  FIG. 3  or as encoded by the cDNA clone selected from the group consisting of cDNA clones contained in Gen Bank Accession No. AF023847, AF1544420, AF154421, AF020390, AF154423, AF154424 and AF154422; and    (c) a nucleotide sequence complementary to any of the nucleotide sequences in (a). or (b), above.    
     
     
         23 . The nucleic acid molecule of  claim 22  wherein said polynucleotide has a complete nucleotide sequence in  FIG. 2  selected from the group consisting of SEQ ID NOs: 1-3 and 5-7.  
     
     
         24 . The nucleic acid molecule of  claim 22  wherein said polynucleotide has a nucleotide sequence in  FIG. 2  selected from the group consisting of SEQ ID NOs: 1-3 and 5-7 encoding the β-galactosidase polypeptide having the complete amino acid sequence designated TBG1-3 and 5-7, respectively.  
     
     
         25 . The nucleic acid molecule of  claim 22  wherein said polynucleotide has the nucleotide sequence in  FIG. 2  selected from the group consisting of SEQ ID NOs: 1-3 and 5-7encoding the mature polypeptide having the amino acid sequence designated TBG1-3 and 5-7, respectively.  
     
     
         26 . The nucleic acid molecule of  claim 22  wherein said polynucleotide has the complete nucleotide sequence of the cDNA clone contained in an Gen Bank Accession No. selected from the group consisting of ATCC Deposit No. selected from the group consisting of AF023847, AF1544420, AF154421, AF020390, AF154423, AF154424 and AF154422.  
     
     
         27 . A method of modifying cell wall metabolism in a plant which comprises transforming said plant with a DNA construct adapted to modify the activity of a β-galactosidase, growing said plant or its descendent and selecting a plant having modified cell wall characteristics, said construct comprising a transcriptional initiation region operative in plants operably linked to a DNA sequence encoding at least one β-galactosidase.  
     
     
         28 . A method as claimed in  claim 27 , wherein said DNA sequence is selected from the group consisting of the sequences of nucleic acid molecules claimed in  claim 1  or  claim 22 .  
     
     
         29 . A plant cell transformed with a nucleic acid molecule as claimed in  claim 1  or  claim 22 .  
     
     
         30 . A plant derived from a plant cell as claimed in  claim 29 .  
     
     
         31 . A plant seed derived from a plant as claimed in  claim 30 .  
     
     
         32 . A method for modifying β-galactosidase gene expression in a plant comprising transforming said plant with a nucleic acid molecule as claimed in  claim 1  or  claim 22 , growing the transformed plant and selecting a plant having modified β-galactosidase gene expression when compared with an untransformed plant.