Genes coding for tomato beta-galactosidase polypeptides

a technology of beta-galactosidase and polypeptides, which is applied in the field of genes coding for tomato beta-galactosidase polypeptides, can solve the problem of elusive cloning of the corresponding gen

Inactive Publication Date: 2005-01-20
UNITED STATES OF AMERICA AS REPRESENTED BY THE SEC OF AGRI THE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent describes an enzyme that breaks down a sugar called galactose found in plant cells. This breakdown can trigger the process of ripping, which involves producing more ethylene gas. It's believed that this action helps initiate or speed up the development of fruits and vegetables.

Problems solved by technology

This patent discusses how researchers have studied the process of softening in tomato fruit caused by the breakdown of pectin molecules in the cell wall. The problem this causes is that current methods of controlling pectin content through manipulation of gene expression have proven effective at preventing softening. To address this issue, the patent seeks to clone a new gene for beta-galactosidase II and its associated polypeptides, allowing direct modification of the cell wall and ultimately reducing softening in fruits like tomato.

Method used

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  • Genes coding for tomato beta-galactosidase polypeptides
  • Genes coding for tomato beta-galactosidase polypeptides
  • Genes coding for tomato beta-galactosidase polypeptides

Examples

Experimental program
Comparison scheme
Effect test

example 1

RNA Extraction

[0081] Tomato (Lycopersicon esculentum Mill., cv. ‘rutgers’) plants were grown in a greenhouse using standard cultural practices. The ripening mutants, ripening inhibitor (rin), non-ripening (nor) and never ripe (Nr) (Tigchelaar et al. 1978. Hort. Science 13: 508-513), were all in the ‘Rutgers’ background. Flowers were tagged at anthesis and fruit were harvested according to the number of days post-anthesis (dpa) or based on their surface color using ripeness stages as previously described (Mitcham et al. 1989. Plant Physiol. 89: 477-481), the complete disclosure of which is hereby fully incorporated herein by reference. For gene expression studies, a variety of leaf, flower, and stem tissues were harvested from greenhouse-grown plants and roots were harvested from seedlings grown in basal tissue culture medium for 4 weeks after seed germination.

[0082] Fruits were processed immediately after harvest in the greenhouse by chilling on ice, excising the various tissues a...

example 2

RT-PCR

[0083] Degenerate primers were designed based on the highest shared deduced amino acid sequence identity we found between an apple (accession number P48980), asparagus (P45582) and carnation (Q00662) β-galactosidase cDNA clones. The two primers used for the first reaction were BG5′E1 (WSNGGNWSNATHCAYTAYCC) and BG3′E (CCRTAYTCRTCNADNGGNGG). A second reaction was done on the products of the first reaction using BG5′I1 (ATHCARACNTAYGTNTTYTGG) and BG3′E. The degeneracy code for the primer sequences is N=a+t+c+g; H=a+t+c; B=t+c+g; D=a+t+g; V=a+c+g; R=a+g; Y=c+t; M=a+c; K=t+g; S=c+g; and W=a+t. The 5′ and 3′ primers corresponded to amino acids 72-78 and 321-315 of the apple clone, respectively. Amplification was done using AmpliTaq DNA polymerase (Perkin Elmer, Norwalk, Conn.) and standard PCR conditions using the cDNA made for the first cDNA library described below as a template (Ausubel et al. 1987. In: Current Protocols in Molecular Biology, John Wiley and Sons, New York, N.Y.)....

example 3

cDNA Library Construction

[0084] Two cDNA libraries were constructed. The first comprised poly(A) RNA isolated from breaker, turning and pink fruit pericarp from ‘Rutgers’ plants. The cDNA synthesis and library construction was done exactly according to the manufacturers instructions for the ZAP-cDNA Gigapack II Gold Cloning Kit (Stratagene), the complete disclosure of which is fully incorporated herein by reference. First-strand cDNA synthesis was primed using a poly(dT) primer and inserts were directionally cloned into the Uni-Zap XR vector using EcoRI and XhoI restriction sites. The second library comprised poly(A) RNA isolated from all fruit tissues (except seeds) from immature green, mature green, breaker, turning, pink, red-ripe and over-ripe fruit of ‘Rutgers’ plants. The cDNA synthesis and library construction was done exactly according to the manufacturers instructions for the SuperScript Lambda System for cDNA synthesis and Cloning (Gibco BRL, Gaithersburg, Md.). First-str...

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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.

Description

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Claims

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Application Information

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Owner UNITED STATES OF AMERICA AS REPRESENTED BY THE SEC OF AGRI THE
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