U.S. patent application number 13/031636 was filed with the patent office on 2011-06-09 for polynucleotides and polypeptides encoded therefrom and methods of using same for increasing biomass in plants and plants generated thereby.
This patent application is currently assigned to The State of Israel, Ministry of Agriculture & Rural Development, Agricultural Research. Invention is credited to Nir Dai, Marina Petreikov, Arthur A. SCHAFFER.
Application Number | 20110138500 13/031636 |
Document ID | / |
Family ID | 38920481 |
Filed Date | 2011-06-09 |
United States Patent
Application |
20110138500 |
Kind Code |
A1 |
SCHAFFER; Arthur A. ; et
al. |
June 9, 2011 |
POLYNUCLEOTIDES AND POLYPEPTIDES ENCODED THEREFROM AND METHODS OF
USING SAME FOR INCREASING BIOMASS IN PLANTS AND PLANTS GENERATED
THEREBY
Abstract
A method of increasing biomass, vigor and/or yield of a plant is
disclosed. The method comprises expressing within the plant an
exogenous polypeptide comprising a UGGPase activity. The
polypeptide may comprise an amino acid sequence at least 90%
homologous, and/or at least 80% identical to SEQ ID NO: 33 as
determined using the BlastP software of the National Center of
Biotechnology Information (NCBI) using default parameters.
Polynucleotides encoding same and plants expressing same are also
disclosed.
Inventors: |
SCHAFFER; Arthur A.;
(Hashmonaim, IL) ; Dai; Nir; (Kiryat-Ono, IL)
; Petreikov; Marina; (Rishon-LeZion, IL) |
Assignee: |
The State of Israel, Ministry of
Agriculture & Rural Development, Agricultural Research
Beit-Dagan
IL
Organization, (A.R.O.), Volcani Center
|
Family ID: |
38920481 |
Appl. No.: |
13/031636 |
Filed: |
February 22, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11822256 |
Jul 3, 2007 |
7906705 |
|
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13031636 |
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60817687 |
Jul 3, 2006 |
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Current U.S.
Class: |
800/290 ;
435/410; 536/23.2; 800/278 |
Current CPC
Class: |
C12N 15/8241 20130101;
C12N 9/1241 20130101 |
Class at
Publication: |
800/290 ;
435/410; 800/278; 536/23.2 |
International
Class: |
C12N 15/82 20060101
C12N015/82; C12N 5/04 20060101 C12N005/04; C12N 15/83 20060101
C12N015/83; C07H 21/00 20060101 C07H021/00 |
Claims
1. A plant cell comprising an exogenous polypeptide comprising a
UGGPase activity.
2. The plant cell of claim 1, wherein said plant cell forms a part
of a plant.
3. The plant cell of claim 1, wherein said exogenous polypeptide
has an amino acid sequence at least 90% homologous, and/or at least
80% identical to SEQ ID NO: 33 as determined using the BlastP
software of the National Center of Biotechnology Information (NCBI)
using default parameters.
4. The plant cell of claim 1, wherein said UGGPase activity
comprises a higher affinity for a Glucose-1-phosphate than a
Galactose-1-phophate.
5. The plant cell of claim 1, wherein a maximum enzyme velocity
(V.sub.max) of said UGGPase activity is higher for a
Galactose-1-phosphate than a Glucose-1-phosphate.
6. The plant cell of claim 1, wherein said exogenous polypeptide
comprises a higher enzymatic activity towards a galactose substrate
than an enzymatic activity of a UGPase for a galactose
substrate.
7. The plant cell of claim 1, wherein said polypeptide is capable
of converting Gal-1-phosphate to UDP-Gal and further is capable of
converting UDP-glucose to glucose-1-phosphate.
8. The plant cell of claim 1, further comprising an exogenous
UGPase.
9. The plant cell of claim 8, wherein said exogenous UGpase
comprises an amino acid sequence as set forth in SEQ ID NO: 35.
10. A method of increasing biomass, vigor and/or yield of a plant
comprising expressing within the plant an exogenous polypeptide
comprising a UGGPase activity, thereby increasing biomass, vigor
and/or yield of the plant.
11. The method of claim 10, wherein said exogenous polypeptide
comprises an amino acid sequence at least 90% homologous, and/or at
least 80% identical to SEQ ID NO: 33 as determined using the BlastP
software of the National Center of Biotechnology Information (NCBI)
using default parameters.
12. The method of claim 10, wherein said expressing is effected by
introducing to said plant a nucleic acid construct which comprise a
polynucleotide sequence encoding said polypeptide and at least one
promoter capable of directing transcription of said polynucleotide
in said plant cell.
13. The method of claim 12, wherein said at least one promoter is a
constitutive promoter.
14. The method of claim 12, wherein said at least one promoter is
an inducible promoter.
15. The method of claim 12, wherein said expressing is effected by
infecting said plant with a virus.
16. The method of claim 15, wherein said virus is an avirulent
virus.
17. The method of claim 10, further comprising expressing within
the plant an exogenous UGPase.
18. An isolated polynucleotide comprising a nucleic acid sequence
encoding a polypeptide having an amino acid sequence at least 90%
homologous, and/or at least 80% identical to SEQ ID NO: 33 as
determined using the BlastP software of the National Center of
Biotechnology Information (NCBI) using default parameters, wherein
said polypeptide comprises a UDP glucose/galactose
pyrophosphorylase (UGGPase) activity.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 11/822,256 filed on Jul. 3, 2007, which claims
the benefit of priority of U.S. Provisional Patent Application No.
60/817,687 filed on Jul. 3, 2006. The contents of the above
Applications are all incorporated herein by reference.
FIELD AND BACKGROUND OF THE INVENTION
[0002] The present invention relates to methods of increasing
biomass in plants and plants generated thereby.
[0003] Plants specifically improved for agriculture, horticulture,
biomass conversion, and other industries (e.g. paper industry,
plants as production factories for proteins or other compounds) can
be obtained using molecular technologies.
[0004] Availability and maintenance of a reproducible stream of
food and animal feed to feed animals and people has been a high
priority throughout the history of human civilization and lies at
the origin of agriculture. Specialists and researchers in the
fields of agronomy science, agriculture, crop science,
horticulture, and forest science are even today constantly striving
to find and produce plants with an increased growth potential to
feed an increasing world population and to guarantee a supply of
reproducible raw materials. The robust level of research in these
fields of science indicates the level of importance leaders in
every geographic environment and climate around the world place on
providing sustainable sources of food, feed, chemicals and energy
for the population.
[0005] Manipulation of crop performance has been accomplished
conventionally for centuries through plant breeding. The breeding
process is, however, both time-consuming and labor-intensive.
Furthermore, appropriate breeding programs must be specially
designed for each relevant plant species.
[0006] On the other hand, great progress has been made in using
molecular genetic approaches to manipulate plants to provide better
crops. Through introduction and expression of recombinant nucleic
acid molecules in plants, researchers are now poised to provide the
community with plant species tailored to grow more efficiently and
produce more product despite unique geographic and/or climatic
environments. These new approaches have the additional advantage of
not being limited to one plant species, but instead being
applicable to multiple different plant species (Zhang et al. (2004)
Plant Physiol. 135:615).
[0007] Despite this progress, today there continues to be a great
need for generally applicable processes that improve forest or
agricultural plant growth to suit particular needs depending on
specific environmental conditions.
[0008] Cellulose, the most abundant organic polymer in the world,
is deposited in the stems of plants and is extensively utilized for
fuel, timber, forage, fibre and chemical cellulose.
[0009] Cellulose synthesis, in contrast with starch, is essentially
an irreversible sink. Cellulose is produced from the precursor
UDP-glucose, which can be formed via two potential pathways.
UDP-glucose can be derived from the cleavage of sucrose in a
reaction catalyzed by sucrose synthase (SuSy; EC 2.4.1.13) yielding
UDP-glucose and fructose. Alternatively, UDP-glucose can be
generated from the phosphorylation of glucose-1-phosphate in a
reaction catalyzed by UDP-glucose pyrophosphorylase (UGPase, EC
2.7.7.9).
[0010] Another potential source of UDP-glucose is galactose. The
entry of free galactose into metabolism begins with its
phosphorylation by galactokinase (EC 2.7.1.6) to Gal-1-P. Following
phosphorylation, two alternative pathways exist for the fate of the
Gal-1-P in plants. One pathway is via the Leloir reaction, carried
out by a uridyltransferase (UT, UDP-Glc: Hexose-1-P
uridyltransferase, EC 2.7.7.12) utilizing UDP-Glc in a transferase
reaction. However, this enzyme is generally not observed in most
plants.
[0011] In an alternative pathway, Gal-1-P may be converted into
UDP-Gal via a pyrophosphorylase (PPase, Gal-1-P: UTP transferase)
utilizing UTP:
Gal-1-P+UTP.rarw..fwdarw.PPi+UDP-Gal.
[0012] The UDP-Gal product of this pathway is further metabolized
to UDP-Glc via the epimerase reaction.
[0013] Previous studies have shown that the melon fruit, with its
active Gal metabolism, shows little UT activity, suggesting that a
PPase is responsible for Gal-1-P metabolism [Smart and Pharr, 1981,
Planta 153: 370-375; Feusi et al., 1999, Physiol Plant 106: 9-16].
There is no known PPase that is specific for the Gal moiety in
melon fruit [Smart and Pharr, 1981, Planta 153: 370-375; Feusi et
al., 1999, Physiol Plant 106: 9-16]. Rather, there appears to be a
PPase in melon fruit which can utilize both Gal-1-P and Glc-1-P.
This dual substrate PPase is present in cucurbit fruit in addition
to the UGPase (UDP-Glc PPase, E.C. 2.7.7.9) which is specific for
the Glc-1-P sugar, and inactive with Gal-1-P [Smart and Pharr,
1981, Planta 153: 370-375; Feusi et al., 1999, Physiol Plant 106:
9-16; Gao et al., 1999, Physiol Plant 106: 1-8]. Feusi et al.
(1999) purified and characterized an enzyme fraction from melon
fruit which catalyzed the nucleotide transfer to both Glc-1-P and
Gal-1-P and were unable to further separate the activities,
suggesting that the two reactions are catalyzed by the same protein
(a UGGPase).
[0014] A UGGPase enzyme was described in germinating pea seeds
(Kotake et al., 2004, J Biol. Chem. 2004 Oct. 29;
279(44):45728-36). The enzyme catalyzed the formation of UDP-Glc,
UDP-Gal, UDP-glucuronic acid, UDP-1-arabinose, and UDP-xylose from
respective monosaccharide 1-phosphates in the presence of UTP as a
co-substrate, indicating that the enzyme has broad substrate
specificity toward monosaccharide 1-phosphates.
[0015] It has been shown that there is a correlation between plant
cellulose content and overall biomass. For example, a gene for
UDP-glucose pyrophosphorylase has been cloned, and sense constructs
inserted in tobacco plants. Heightened enzyme activity and
cellulose synthesis were reported [Xue et al. 1997, Plant Physiol.
114(suppl 3):300]. Analyses indicated a 30% enhancement of
cellulose content and a 20% increase in biomass. In addition,
Coleman et al [Plant Biotechnology Journal. 4: 87-101, 2006] teach
transgenic expression of UDP-Glc PPase in aspen trees and show a
significant increase in plant height and biomass.
[0016] There is thus a widely recognized need for, and it would be
highly advantageous to identify novel enzymes which utilize both
glucose and galactose substrates for increasing cellulose content
and biomass in plants.
SUMMARY OF THE INVENTION
[0017] According to one aspect of the present invention there is
provided an isolated polynucleotide comprising a nucleic acid
sequence encoding a polypeptide having an amino acid sequence at
least 90% homologous, and/or at least 80% identical to SEQ ID NO:
33 as determined using the BlastP software of the National Center
of Biotechnology Information (NCBI) using default parameters,
wherein the polypeptide comprises a UDP glucose/galactose
pyrophosphorylase (UGGPase) activity.
[0018] According to another aspect of the present invention there
is provided an isolated polypeptide comprising an amino acid
sequence at least 90% homologous, and/or at least 80% identical to
SEQ ID NO: 33 as determined using the BlastP software of the
National Center of Biotechnology Information (NCBI) using default
parameters, wherein the polypeptide comprises a UGGPase
activity.
[0019] According to yet another aspect of the present invention
there is provided an isolated polynucleotide comprising a nucleic
acid sequence encoding a polypeptide as set forth in SEQ ID NO:
33.
[0020] According to an additional aspect of the present invention
there is provided an isolated polynucleotide comprising a nucleic
acid sequence as set forth in SEQ ID NO: 34.
[0021] According to yet an additional aspect of the present
invention there is provided an isolated polynucleotide comprising a
nucleic acid sequence encoding a polypeptide having an amino acid
sequence at least 95% homologous, and/or at least 90% identical to
SEQ ID NO: 35 as determined using the BlastP software of the
National Center of Biotechnology Information (NCBI) using default
parameters, wherein the polypeptide comprises a UDP glucose
pyrophosphorylase (UGPase) activity.
[0022] According to still an additional aspect of the present
invention there is provided an isolated polynucleotide comprising a
nucleic acid sequence encoding a polypeptide as set forth in SEQ ID
NO: 35.
[0023] According to a further aspect of the present invention there
is provided an isolated polynucleotide comprising a nucleic acid
sequence as set forth in SEQ ID NO: 36.
[0024] According to yet a further aspect of the present invention
there is provided an isolated polypeptide comprising an amino acid
sequence at least 95% homologous, and/or at least 90% identical to
SEQ ID NO: 35 as determined using the BlastP software of the
National Center of Biotechnology Information (NCBI) using default
parameters, wherein the polypeptide comprises a UGPase
activity.
[0025] According to still a further aspect of the present invention
there is provided an isolated polypeptide comprising an amino acid
sequence as set forth in SEQ ID NO: 35.
[0026] According to still a further aspect of the present invention
there is provided an isolated polynucleotide comprising a nucleic
acid sequence encoding a polypeptide having an amino acid sequence
at least 85% homologous, and/or at least 75% identical to SEQ ID
NO: 37 as determined using the BlastP software of the National
Center of Biotechnology Information (NCBI) using default
parameters, wherein the polypeptide comprises a uridyltransferase
(UT) activity.
[0027] According to still a further aspect of the present invention
there is provided an isolated polynucleotide comprising a nucleic
acid sequence encoding a polypeptide as set forth in SEQ ID NO:
37.
[0028] According to still a further aspect of the present invention
there is provided an isolated polynucleotide comprising a nucleic
acid sequence as set forth in SEQ ID NO: 38.
[0029] According to still a further aspect of the present invention
there is provided an isolated polypeptide comprising an amino acid
sequence at least 85% homologous, and/or at least 75% identical to
SEQ ID NO: 37 as determined using the BlastP software of the
National Center of Biotechnology Information (NCBI) using default
parameters, wherein the polypeptide comprises a UT activity.
[0030] According to still a further aspect of the present invention
there is provided an isolated polypeptide comprising an amino acid
sequence as set forth in SEQ ID NO: 38.
[0031] According to still a further aspect of the present invention
there is provided a plant cell comprising an exogenous polypeptide
comprising an amino acid sequence at least 90% homologous, and/or
at least 80% identical to SEQ ID NO: 33 as determined using the
BlastP software of the National Center of Biotechnology Information
(NCBI) using default parameters, wherein the polypeptide comprises
a UGGPase activity.
[0032] According to yet a further aspect of the present invention
there is provided a plant cell comprising an exogenous polypeptide
comprising an amino acid sequence at least 95% homologous, and/or
at least 90% identical to SEQ ID NO: 35 as determined using the
BlastP software of the National Center of Biotechnology Information
(NCBI) using default parameters, wherein the polypeptide comprises
a UGPase activity.
[0033] According to still a further aspect of the present invention
there is provided a plant cell comprising an exogenous polypeptide
comprising an amino acid sequence at least 85% homologous, and/or
at least 75% identical to SEQ ID NO: 37 as determined using the
BlastP software of the National Center of Biotechnology Information
(NCBI) using default parameters, wherein the polypeptide comprises
a UT activity.
[0034] According to still a further aspect of the present invention
there is provided a method of increasing biomass, vigor and/or
yield of a plant comprising expressing within the plant an
exogenous polypeptide comprising a UGGPase activity, thereby
increasing biomass, vigor and/or yield of the plant.
[0035] According to further features in preferred embodiments of
the invention described below, the UGGPase activity comprises a
higher affinity for a Glucose-1-phosphate than a
Galactose-1-phophate.
[0036] According to still further features in the described
preferred embodiments, the isolated polypeptide comprises a Km for
Galactose-1-phosphate of about 0.43 mM.
[0037] According to still further features in the described
preferred embodiments, the isolated polypeptide comprises a Km for
Glucose-1-phosphate of about 0.27 mM.
[0038] According to still further features in the described
preferred embodiments, the isolated polypeptide comprises a Km for
UDP-Galactose of about 0.44 mM.
[0039] According to still further features in the described
preferred embodiments, the isolated polypeptide comprises a Km for
UDP-Glucose of about 0.14 mM.
[0040] According to still further features in the described
preferred embodiments, a maximum enzyme velocity (V.sub.max) of the
UGGPase activity is higher for a Galactose-1-phosphate than a
Glucose-1-phosphate.
[0041] According to still further features in the described
preferred embodiments, the isolated polypeptide comprises a Vmax
for Galactose-1-phosphate of about 714 .mu.mol mg protein.sup.-1
min.sup.-1.
[0042] According to still further features in the described
preferred embodiments, the isolated polypeptide comprises a Vmax
for Glucose-1-phosphate of about 222 .mu.mol mg protein.sup.-1
min.sup.-1.
[0043] According to still further features in the described
preferred embodiments, the isolated polypeptide comprises a Vmax
for UDP-Galactose of about 625 .mu.mol mg protein.sup.-1
min.sup.-1.
[0044] According to still further features in the described
preferred embodiments, the isolated polypeptide comprises a Vmax
for UDP-Glucose of about 238 .mu.mol mg protein.sup.-1
min.sup.-1.
[0045] According to still further features in the described
preferred embodiments, the isolated polypeptide comprises a higher
enzymatic activity towards a galactose substrate than an enzymatic
activity of a UGPase for a galactose substrate.
[0046] According to still further features in the described
preferred embodiments, the polypeptide is capable of converting
Gal-1-phosphate to UDP-Gal and further is capable of converting
UDP-glucose to glucose-1-phosphate.
[0047] According to still further features in the described
preferred embodiments, the isolated polypeptide comprises an amino
acid sequence as set forth by SEQ ID NO: 33.
[0048] According to still further features in the described
preferred embodiments, the plant cell forms a part of a plant.
[0049] According to still further features in the described
preferred embodiments, the plant further comprises an exogenous
UGPase.
[0050] According to still further features in the described
preferred embodiments, the exogenous UGpase comprises an amino acid
sequence as set forth in SEQ ID NO: 35.
[0051] According to still further features in the described
preferred embodiments, the plant cell forms a part of a plant.
[0052] According to still further features in the described
preferred embodiments, the exogenous polypeptide comprises an amino
acid sequence at least 90% homologous, and/or at least 80%
identical to SEQ ID NO: 33 as determined using the BlastP software
of the National Center of Biotechnology Information (NCBI) using
default parameters.
[0053] According to still further features in the described
preferred embodiments, the expressing is effected by introducing to
the plant a nucleic acid construct which comprises a polynucleotide
sequence encoding the polypeptide and at least one promoter capable
of directing transcription of the polynucleotide in the plant
cell.
[0054] According to still further features in the described
preferred embodiments, the at least one promoter is a constitutive
promoter.
[0055] According to still further features in the described
preferred embodiments, the at least one promoter is an inducible
promoter.
[0056] According to still further features in the described
preferred embodiments, the expressing is effected by infecting the
plant with a virus.
[0057] According to still further features in the described
preferred embodiments, the virus is an avirulent virus.
[0058] According to still further features in the described
preferred embodiments, the method further comprises expressing
within the plant an exogenous UGPase.
[0059] The present invention successfully addresses the
shortcomings of the presently known configurations by providing
polypeptides and polynucleotides encoding same, capable of
upregulating plant growth and yield.
[0060] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In case of conflict, the patent specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
BRIEF DESCRIPTION OF THE DRAWINGS
[0061] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0062] The invention is herein described, by way of example only,
with reference to the accompanying drawings. With specific
reference now to the drawings in detail, it is stressed that the
particulars shown are by way of example and for purposes of
illustrative discussion of the preferred embodiments of the present
invention only, and are presented in the cause of providing what is
believed to be the most useful and readily understood description
of the principles and conceptual aspects of the invention. In this
regard, no attempt is made to show structural details of the
invention in more detail than is necessary for a fundamental
understanding of the invention, the description taken with the
drawings making apparent to those skilled in the art how the
several forms of the invention may be embodied in practice.
[0063] In the drawings:
[0064] FIG. 1A depicts partial purification of melon fruit UGGPase
(SEQ ID NO: 33) on HPLC-MonoQ. Closed circles indicate activity
with UDP-Glc in the pyrophosphorolytic direction and open circles
indicate activity with Gal-1-P in the synthesis direction.
[0065] FIG. 1B depicts a Western blot using UGGPase antibodies
(Feusi et al., 1999) of electrophoretic separations of MW markers
(first lane), crude melon fruit extracts (second lane) and HPLC
MonoQ fractions exhibiting UGGPase activity (third lane); C,
Coomassie Blue protein stain of the HPLC MonoQ fractions exhibiting
UGGPase activity: MW markers (first lane) and HPLC MonoQ fractions
exhibiting UGGPase activity (second lane). The band in the
Coomassie stain, indicated by the arrow and corresponding to the
band in the immunoblot, was excised and microsequenced.
[0066] FIGS. 2A-C are protein sequence homology alignments of plant
UGGPase (SEQ ID NO: 33), UGPase (SEQ ID NO: 35) and UT (SEQ ID NO:
37). Sequences in bold indicate the seven peptide sequences (SEQ ID
NOs: 39-45) obtained from the peptide microsequencing of the
purified protein. Underlined sequences indicate conserved sequences
used for the preparation of degenerate primers for the PCR cloning
of the melon genes. Accession numbers of the sequence presented
are: UGGPase: melon (DQ399739), Arabidopsis (AF360236), pea
(AB178642); UT: melon DQ445484, potato (TC28197), Arabidopsis
(NM.sub.--121825); UGPase: melon DQ445483, Arabidopsis
(NM.sub.--121737), potato (U20345), barley (Q07131).
[0067] FIGS. 3A-C is a Coomassie stained SDS-PAGE gel loaded with
an E. coli protein extract following heterologous expression of
melon UGGPase (SEQ ID NO: 33; FIG. 3A); melon UGPase (SEQ ID NO:
35; FIG. 3B); and melon UT (SEQ ID NO: 37; FIG. 3C). For each
enzyme the three lanes represent, respectively, the MW marker, the
E. coli extract with the expressed protein (+IPTG) and the E. coli
extract without the heterologously expressed protein (-IPTG).
[0068] FIG. 4 is a graph depicting hydrophobic interaction
chromatography separation (HIC, phenyl sepharose) of UGPase (SEQ ID
NO: 35) and UGGPase (SEQ ID NO: 33) from melon fruit ovaries.
Closed circles indicate activity with Gal-1-P and open circles
indicate activity with Glc-1-P. Peak I is the Glc-1-P specific
UGPase and peak II is the UGGPase enzyme.
[0069] FIGS. 5A-B depict expression patterns of UGGPase (SEQ ID NO:
33), UGPase (SEQ ID NO: 35) and UT (SEQ ID NO: 37) in developing
melon fruit by Northern blots (FIG. 5A; UT was not detected and is
not presented) and by Quantitative RT-PCR of UT and UGGPase (FIG.
5B). mRNA expression is relative to the expression of the melon
actin gene. DAA, days after anthesis; rRNA, ribosomal RNA.
[0070] FIG. 6 is a schematic diagram of the proposed pathway of
galactose metabolism in melon fruit, emphasizing the dual role of
the UGGPase. The enzymes involved in galactose metabolism in melon
fruit are represented in italics.
[0071] FIG. 7 is a phylogenetic tree of nucleotide-sugar metabolism
enzymes. The abbreviations and the accession numbers of the
sequences used in the preparation of the tree are as follows: UGP,
UGPase: Homo sapiens (Hs), Q07131; Arabidopsis thaliana (At),
NM.sub.--121737; Cucumis melo (Cm), DQ445483; Solanum tuberosum
(St), U20345. NAGA, UDP-N-acetyl-Gal/Glc amine PPase: Homo sapiens
(Hs), BC009377; Arabidopsis thaliana (At), BT020380; Oryza sativa
(Os), AK071409. UGGP, UGGPase: Oryza sativa (Os), AK064009;
Arabidopsis thaliana (At), AF360236; Pisum sativum (Ps), AB178642;
Cucumis melo (Cm), DQ399739. UT, uridyltransferase: Homo sapiens
(Hs), P07902; Arabidopsis thaliana (At), NM.sub.--121825; Cucumis
melo (Cm), DQ445484; Solanum tuberosum (St), TC28197. AGP, ADPglu
PPase, small subunit: Arabidopsis thaliana (At), NM.sub.--124205;
Citrus unshiu (Cu), AF184597; Cucumis melo (Cm), AF030382; Pisum
sativum (Ps), X96764. The tree was prepared using the Clustal X
alignment and Treeview programs. Bar represents distance value of
0.1 substitution per site.
[0072] FIG. 8 is a photograph illustrating UGGPase (SEQ ID NO: 33)
expression level in independent transgenic plants following
semi-quantitative RT-PCR in 25 and 35 cycles.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0073] The present invention is of polypeptides and polynucleotides
encoding same capable of increasing plant biomass and/or ethanol
production.
[0074] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not limited
in its application to the details set forth in the following
description or exemplified by the Examples. The invention is
capable of other embodiments or of being practiced or carried out
in various ways. Also, it is to be understood that the phraseology
and terminology employed herein is for the purpose of description
and should not be regarded as limiting.
[0075] Cellulose is produced from the precursor UDP-glucose, which
can be formed via a number of enzymatic reactions, including via
the cleavage of sucrose (in a reaction catalyzed by sucrose
synthase (SuSy; EC 2.4.1.13)) and/or the phosphorylation of
glucose-1-phosphate (in a reaction catalyzed by UDP-glucose
pyrophosphorylase (UGPase, EC 2.7.7.9)). Following expression of
UGPase in tobacco plants, heightened enzyme activity and cellulose
synthesis were reported [Xue et al. 1997, Plant Physiol. 114(suppl
3):300].
[0076] Another potential source of UDP-glucose is galactose.
Gal-1-P may be converted into UDP-Gal via a pyrophosphorylase
(PPase, Gal-1-P: UTP transferase) utilizing UTP:
Gal-1-P+UTP.rarw..fwdarw.PPi+UDP-Gal.
[0077] The UDP-Gal product of this pathway is further metabolized
to UDP-Glc via the epimerase reaction.
[0078] Through meticulous experimentation, the present inventors
cloned and sequenced the melon UGGPase (FIGS. 1-2A). This enzyme is
capable of utilizing both glucose and galactose as a source of
starting material for generating cellulose and showed that it was
possible to induce the expression thereof in tobacco cell plants
(FIG. 8).
[0079] In addition, the present inventors cloned and sequenced for
the first time, melon UGPase (FIG. 2B) and melon uridyltransferase
(UT; FIG. 2C), two enzymes which, in conjunction with expressing
the UGGPase of the present invention, may further aid in increasing
cellulose biomass.
[0080] The three melon enzymes were bacterially expressed (FIGS.
3A-C) and characterized (FIGS. 5A-B and Tables 1-5).
[0081] Thus, according to one aspect of the present invention there
is provided a method of increasing biomass, vigor and/or yield of a
plant comprising expressing within the plant an exogenous
polypeptide comprising a UGGPase activity. The present invention
also contemplates expression of other homologues, orthologues and
active portions of the above mentioned exogenous polypeptides as
will be further described hereinbelow.
[0082] As used herein the phrase "plant biomass" refers to the
amount or quantity of tissue (in particular cellulose comprising
tissue) produced from the plant in a growing season, which could
also determine or affect the plant yield or the yield per growing
area.
[0083] As used herein the phrase "plant vigor" refers to the amount
or quantity of (cellulose comprising) tissue produced from the
plant in a given time. Hence increase vigor could determine or
affect the plant yield or the yield per growing time or growing
area.
[0084] As used herein the phrase "plant yield" refers to the amount
or quantity of (cellulose comprising) tissue produced and harvested
as the plant produced product. Hence increase yield could affect
the economic benefit one can obtain from the plant in a certain
growing time.
[0085] Methods of determining biomass, yield and vigor are well
known in the art and further described in Coleman et al, 2006,
Plant Biotechnology Journal 4 (1), 87-101.
[0086] As used herein the term "improving" or "increasing" refers
to improving or increasing the biomass/yield/vigor of the
transgenic plant of the present invention by at least about 2%
more, 5% more, 10% more, 20% more, 30% more, 40% more, 50% more,
60% more, 70% more, 80% more, 90% or more than that of the
non-transgenic plant (e.g., mock transfected, or naive).
[0087] The term "plant" as used herein encompasses whole plants,
ancestors and progeny of the plants and plant parts, including
seeds, shoots, stems, roots (including tubers), and plant cells,
tissues and organs. The term "plant" also therefore encompasses
suspension cultures, embryos, meristematic regions, callus tissue,
leaves, gametophytes, sporophytes, pollen, and microspores. Plants
that are particularly useful in the methods of the invention
include all plants which belong to the superfamily Viridiplantae,
in particular monocotyledonous and dicotyledonous plants which are
of commercial value, including a fodder or forage legume,
ornamental plant, food crop, tree, or shrub selected from the
following non-limiting list comprising maize, sweet potato, tubers
such as cassarva, sugar beet, wheat, barely, rye, oat, rice,
soybean, peanut, pea, lentil and alfalfa, cotton, rapeseed, canola,
pepper, sunflower, potato, tobacco, tomato, eggplant, trees such as
eucalyptus and poplars, an ornamental plant, a perennial grass and
a forage crop.
[0088] As used herein, the term "exogenous polypeptide" refers to a
polypeptide that is introduced into a cell by artifice, such that a
level of expression thereof is greater than in an identical cell
not comprising the exogenous polypeptide. The expressing is
typically effected by introducing an exogenous polynucleotide
encoding the polypeptide of the present invention into the plant
either in a stable or transient manner as further described herein
below.
[0089] As used herein, the phrase "UGGPase activity" refers to an
enzyme capable of catalyzing the following two reactions:
Gal-1-P+UTP.fwdarw.PPi+UDP-Gal.
Glu-1-P+UTP.fwdarw.PPi+UDP-Glu.
[0090] According to one embodiment of this aspect of the present
invention, the polypeptides of the present invention are also
capable of catalyzing the reverse reactions, as follows:
Gal-1-P+UTP.rarw.PPi+UDP-Gal.
Glu-1-P+UTP.rarw.PPi+UDP-Glu.
[0091] According to one embodiment, the UGGPase activity (EC
2.7.7.64) of the polypeptide of the present invention comprises a
higher affinity for a Glucose-1-phosphate than a
Galactose-1-phophate.
[0092] Methods of determining protein affinity are well known in
the art [e.g., BiaCore and/or Scatchard analyses (RIA)]. An
exemplary method for determining the relative affinity for the
substrates of the polypeptide of the present invention is described
in the general materials and methods section herein below.
[0093] Thus, according to one embodiment, the concentration of
Galactose-1-phosphate that leads to half-maximal velocity (Km) of
the polypeptide of the present invention is about 0.43 mM.
[0094] According to another embodiment, the Km of the polypeptide
of the present invention for Glucose-1-phosphate is about 0.27
mM.
[0095] According to still another embodiment, the Km of the
polypeptide of the present invention for UDP-Galactose is about
0.44 mM.
[0096] According to yet another embodiment, the Km of the
polypeptide of the present invention for UDP-Glucose is about 0.14
mM.
[0097] According to one embodiment, the polypeptide of the present
invention comprises a higher enzymatic activity towards a galactose
substrate (i.e. galactose-1-phosphate) than an enzymatic activity
of a UGPase for a galactose substrate.
[0098] Methods of determining the enzymatic activity of the
polypeptides of the present invention towards their
glucose/galactose substrates are described in the materials and
methods section herein below.
[0099] According to another embodiment, the maximum enzyme velocity
(V.sub.max) of the UGGPase activity of the polypeptide of the
present invention is higher for a Galactose-1-phosphate than a
Glucose-1-phosphate.
[0100] According to yet another embodiment, the Vmax for
Galactose-1-phosphate of the polypeptide of the present invention
is about 714 .mu.mol mg protein.sup.-1 min.sup.-1.
[0101] According to still another embodiment, the Vmax for
Glucose-1-phosphate of the polypeptide of the present invention is
about 222 .mu.mol mg protein.sup.-1 min.sup.-1.
[0102] According to yet another embodiment, the Vmax for
UDP-Galactose of the polypeptide of the present invention is about
625 .mu.mol mg protein.sup.-1 min.sup.-1.
[0103] According to still another embodiment, the Vmax for
UDP-Glucose of the polypeptide of the present invention is about
238 .mu.mol mg protein.sup.-1 min.sup.-1.
[0104] It will be appreciated that the method of increasing biomass
of a plant may be effected by expressing any exogenous UGGPase in a
plant including but not limited to the UGGPase isolated from pea
seeds (Kotake et al., 2004, J Biol Chem. 2004 Oct. 29;
279(44):45728-36). The present inventors searched the EST databases
(www.tigr.org) and showed that other plant families also express
homologues of UGGPase. Such families include, but are not limited
to Solanaceae (tomato, BF05177), Brassicaceae (Arabidopsis,
TC262279), Leguminoseae (soya, TC228175), Compositaceae,
(sunflower, TC10097), and Graminae (wheat, TC251010), although
these are described as unknown proteins. Thus the present invention
contemplates artificial expression of these proteins to increase
biomass of a plant.
[0105] According to one embodiment of this aspect of the present
invention, the exogenous polypeptide comprises an amino acid
sequence at least 90% homologous, and/or at least 80% identical to
SEQ ID NO: 33, which comprises a UGGPase activity. According to one
embodiment the polypeptide comprises an amino acid sequence as set
forth by SEQ ID NO: 33.
[0106] The present invention contemplates expression of any
polynucleotide encoding a polypeptide with an amino acid sequence
at least 90% homologous, and/or at least 80% identical to SEQ ID
NO: 33. For example, the present invention contemplates expression
of a polynucleotide of a sequence as set forth in SEQ ID NO: 34
encoding the polypeptide as set for the in SEQ ID NO: 33.
[0107] Thus, the a nucleic acid sequence is at least about 70%, at
least about 75%, at least about 80%, at least about 81%, at least
about 82%, at least about 83%, at least about 84%, at least about
85%, at least about 86%, at least about 87%, at least about 88%, at
least about 89%, at least about 90%, at least about 91%, at least
about 92%, at least about 93%, at least about 93%, at least about
94%, at least about 95%, at least about 96%, at least about 97%, at
least about 98%, at least about 99%, or more say 100% identical to
a nucleotide sequence selected from the group consisting of SEQ ID
NO: 34.
[0108] Nucleic acid sequences may encode polypeptide sequences
comprising an amino acid sequence at least about 70%, at least
about 75%, at least about 80%, at least about 81%, at least about
82%, at least about 83%, at least about 84%, at least about 85%, at
least about 86%, at least about 87%, at least about 88%, at least
about 89%, at least about 90%, at least about 91%, at least about
92%, at least about 93%, at least about 93%, at least about 94%, at
least about 95%, at least about 96%, at least about 97%, at least
about 98%, at least about 99%, or more say 100% homologous to SEQ
ID NO 33.
[0109] Homology (e.g., percent homology) can be determined using
any homology comparison software, including for example, the BlastP
software of the National Center of Biotechnology Information (NCBI)
such as by using default parameters (as detailed above).
[0110] Identity (e.g., percent homology) can be determined using
any homology comparison software, including for example, the BlastN
software of the National Center of Biotechnology Information (NCBI)
such as by using default parameters (as detailed above).
[0111] According to one preferred embodiment of this aspect of the
present invention the isolated polynucleotide is as set forth in
SEQ ID NO: 34.
[0112] A nucleic acid sequence (also termed herein as isolated
polynucleotide) of the present invention refers to a single or
double stranded nucleic acid sequence which is isolated and
provided in the form of an RNA sequence, a complementary
polynucleotide sequence (cDNA), a genomic polynucleotide sequence
and/or a composite polynucleotide sequences (e.g., a combination of
the above).
[0113] As used herein the phrase "complementary polynucleotide
sequence" refers to a sequence, which results from reverse
transcription of messenger RNA using a reverse transcriptase or any
other RNA dependent DNA polymerase. Such a sequence can be
subsequently amplified in vivo or in vitro using a DNA dependent
DNA polymerase.
[0114] As used herein the phrase "genomic polynucleotide sequence"
refers to a sequence derived (isolated) from a chromosome and thus
it represents a contiguous portion of a chromosome.
[0115] As used herein the phrase "composite polynucleotide
sequence" refers to a sequence, which is at least partially
complementary and at least partially genomic. A composite sequence
can include some exonal sequences required to encode the
polypeptide of the present invention, as well as some intronic
sequences interposing therebetween. The intronic sequences can be
of any source, including of other genes, and typically will include
conserved splicing signal sequences. Such intronic sequences may
further include cis acting expression regulatory elements.
[0116] Nucleic acid sequences of the polypeptides of the present
invention may be optimized for plant expression. Examples of such
sequence modifications include, but are not limited to, an altered
G/C content to more closely approach that typically found in the
plant species of interest, and the removal of codons atypically
found in the plant species commonly referred to as codon
optimization.
[0117] The phrase "codon optimization" refers to the selection of
appropriate DNA nucleotides for use within a structural gene or
fragment thereof that approaches codon usage within the plant of
interest. Therefore, an optimized gene or nucleic acid sequence
refers to a gene in which the nucleotide sequence of a native or
naturally occurring gene has been modified in order to utilize
statistically-preferred or statistically-favored codons within the
plant. The nucleotide sequence typically is examined at the DNA
level and the coding region optimized for expression in the plant
species determined using any suitable procedure, for example as
described in Sardana et al. (1996, Plant Cell Reports 15:677-681).
In this method, the standard deviation of codon usage, a measure of
codon usage bias, may be calculated by first finding the squared
proportional deviation of usage of each codon of the native gene
relative to that of highly expressed plant genes, followed by a
calculation of the average squared deviation. The formula used is:
1 SDCU=n=1N [(Xn-Yn)/Yn]2/N, where Xn refers to the frequency of
usage of codon n in highly expressed plant genes, where Yn to the
frequency of usage of codon n in the gene of interest and N refers
to the total number of codons in the gene of interest. A table of
codon usage from highly expressed genes of dicotyledonous plants is
compiled using the data of Murray et al. (1989, Nuc Acids Res.
17:477-498).
[0118] One method of optimizing the nucleic acid sequence in
accordance with the preferred codon usage for a particular plant
cell type is based on the direct use, without performing any extra
statistical calculations, of codon optimization tables such as
those provided on-line at the Codon Usage Database through the NIAS
(National Institute of Agrobiological Sciences) DNA bank in Japan
(http://www.kazusa.or.jp/codon/). The Codon Usage Database contains
codon usage tables for a number of different species, with each
codon usage table having been statistically determined based on the
data present in Genbank.
[0119] By using the above tables to determine the most preferred or
most favored codons for each amino acid in a particular species
(for example, rice), a naturally-occurring nucleotide sequence
encoding a protein of interest can be codon optimized for that
particular plant species. This is effected by replacing codons that
may have a low statistical incidence in the particular species
genome with corresponding codons, in regard to an amino acid, that
are statistically more favored. However, one or more less-favored
codons may be selected to delete existing restriction sites, to
create new ones at potentially useful junctions (5' and 3' ends to
add signal peptide or termination cassettes, internal sites that
might be used to cut and splice segments together to produce a
correct full-length sequence), or to eliminate nucleotide sequences
that may negatively effect mRNA stability or expression.
[0120] The naturally-occurring encoding nucleotide sequence may
already, in advance of any modification, contain a number of codons
that correspond to a statistically-favored codon in a particular
plant species. Therefore, codon optimization of the native
nucleotide sequence may comprise determining which codons, within
the native nucleotide sequence, are not statistically-favored with
regards to a particular plant, and modifying these codons in
accordance with a codon usage table of the particular plant to
produce a codon optimized derivative. A modified nucleotide
sequence may be fully or partially optimized for plant codon usage
provided that the protein encoded by the modified nucleotide
sequence is produced at a level higher than the protein encoded by
the corresponding naturally occurring or native gene. Construction
of synthetic genes by altering the codon usage is described in for
example PCT Patent Application 93/07278.
[0121] Thus, the present invention encompasses nucleic acid
sequences described hereinabove; fragments thereof, sequences
hybridizable therewith, sequences homologous thereto, sequences
orthologous thereto, sequences encoding similar polypeptides with
different codon usage, altered sequences characterized by
mutations, such as deletion, insertion or substitution of one or
more nucleotides, either naturally occurring or man induced, either
randomly or in a targeted fashion.
[0122] According to an embodiment of this aspect of the present
invention the isolated polypeptide comprises an amino acid sequence
as set forth by SEQ ID NO: 33.
[0123] The present invention also encompasses sequences homologous
and orthologous to the above mentioned polypeptides, fragments of
the above described polypeptides and polypeptides having mutations,
such as deletions, insertions or substitutions of one or more amino
acids, either naturally occurring or man induced, either randomly
or in a targeted fashion.
[0124] Polynucleotides and polypeptides of the present invention
are used for plant expression.
[0125] Expressing the exogenous polynucleotide of the present
invention within the plant can be effected by transforming one or
more cells of the plant with the exogenous polynucleotide, followed
by generating a mature plant from the transformed cells and
cultivating the mature plant under conditions suitable for
expressing the exogenous polynucleotide within the mature
plant.
[0126] Preferably, the transformation is effected by introducing to
the plant cell a nucleic acid construct which includes the
exogenous polynucleotide of the present invention and at least one
promoter capable of directing transcription of the exogenous
polynucleotide in the plant cell. Further details of suitable
transformation approaches are provided hereinbelow.
[0127] As used herein, the term "promoter" refers to a region of
DNA which lies upstream of the transcriptional initiation site of a
gene to which RNA polymerase binds to initiate transcription of
RNA. The promoter controls where (e.g., which portion of a plant,
which organ within an animal, etc.) and/or when (e.g., which stage
or condition in the lifetime of an organism) the gene is
expressed.
[0128] Any suitable promoter sequence can be used by the nucleic
acid construct of the present invention. Preferably the promoter is
a constitutive promoter.
[0129] Suitable constitutive promoters include, for example, CaMV
35S promoter (SEQ ID NO: 46; Odell et al., Nature 313:810-812,
1985); Arabidopsis At6669 promoter (SEQ ID NO: 47); maize Ubi 1
(Christensen et al., Plant Sol. Biol. 18:675-689, 1992) (SEQ ID NO:
48); rice actin (McElroy et al., Plant Cell 2:163-171, 1990) (SEQ
ID NO: 49); pEMU (Last et al., Theor. Appl. Genet. 81:581-588,
1991); and Synthetic Super MAS (Ni et al., The Plant Journal 7:
661-76, 1995) (SEQ ID NO: 50). Other constitutive promoters include
those in U.S. Pat. Nos. 5,659,026, 5,608,149; 5,608,144; 5,604,121;
5,569,597: 5,466,785; 5,399,680; 5,268,463; and 5,608,142.
[0130] Suitable tissue-specific promoters include, but not limited
to, leaf-specific promoters such as described, for example, by
Yamamoto et al., Plant J. 12:255-265, 1997; Kwon et al., Plant
Physiol. 105:357-67, 1994; Yamamoto et al., Plant Cell Physiol.
35:773-778, 1994; Gotor et al., Plant J. 3:509-18, 1993; Orozco et
al., Plant Mol. Biol. 23:1129-1138, 1993; and Matsuoka et al.,
Proc. Natl. Acad. Sci. USA 90:9586-9590, 1993.
[0131] The nucleic acid construct of the present invention
preferably further includes an appropriate selectable marker and/or
an origin of replication. Preferably, the nucleic acid construct
utilized is a shuttle vector, which can propagate both in E. coli
(wherein the construct comprises an appropriate selectable marker
and origin of replication) and be compatible for propagation in
cells. The construct according to the present invention can be, for
example, a plasmid, a bacmid, a phagemid, a cosmid, a phage, a
virus or an artificial chromosome.
[0132] The nucleic acid construct of the present invention can be
utilized to stably or transiently transform plant cells. In stable
transformation, the exogenous polynucleotide of the present
invention is integrated into the plant genome and as such it
represents a stable and inherited trait. In transient
transformation, the exogenous polynucleotide is expressed by the
cell transformed but it is not integrated into the genome and as
such it represents a transient trait.
[0133] There are various methods of introducing foreign genes into
both monocotyledonous and dicotyledonous plants (Potrykus, I.,
Annu. Rev. Plant. Physiol., Plant. Mol. Biol. (1991) 42:205-225;
Shimamoto et al., Nature (1989) 338:274-276).
[0134] The principle methods of causing stable integration of
exogenous DNA into plant genomic DNA include two main
approaches:
[0135] (i) Agrobacterium-mediated gene transfer: Klee et al. (1987)
Annu. Rev. Plant Physiol. 38:467-486; Klee and Rogers in Cell
Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular
Biology of Plant Nuclear Genes, eds. Schell, J., and Vasil, L. K.,
Academic Publishers, San Diego, Calif. (1989) p. 2-25; Gatenby, in
Plant Biotechnology, eds. Kung, S, and Arntzen, C. J., Butterworth
Publishers, Boston, Mass. (1989) p. 93-112.
[0136] (ii) Direct DNA uptake: Paszkowski et al., in Cell Culture
and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of
Plant Nuclear Genes eds. Schell, J., and Vasil, L. K., Academic
Publishers, San Diego, Calif. (1989) p. 52-68; including methods
for direct uptake of DNA into protoplasts, Toriyama, K. et al.
(1988) Bio/Technology 6:1072-1074. DNA uptake induced by brief
electric shock of plant cells: Zhang et al. Plant Cell Rep. (1988)
7:379-384. Fromm et al. Nature (1986) 319:791-793. DNA injection
into plant cells or tissues by particle bombardment, Klein et al.
Bio/Technology (1988) 6:559-563; McCabe et al. Bio/Technology
(1988) 6:923-926; Sanford, Physiol. Plant. (1990) 79:206-209; by
the use of micropipette systems: Neuhaus et al., Theor. Appl.
Genet. (1987) 75:30-36; Neuhaus and Spangenberg, Physiol. Plant.
(1990) 79:213-217; glass fibers or silicon carbide whisker
transformation of cell cultures, embryos or callus tissue, U.S.
Pat. No. 5,464,765 or by the direct incubation of DNA with
germinating pollen, DeWet et al. in Experimental Manipulation of
Ovule Tissue, eds. Chapman, G. P. and Mantell, S. H. and Daniels,
W. Longman, London, (1985) p. 197-209; and Ohta, Proc. Natl. Acad.
Sci. USA (1986) 83:715-719.
[0137] The Agrobacterium system includes the use of plasmid vectors
that contain defined DNA segments that integrate into the plant
genomic DNA. Methods of inoculation of the plant tissue vary
depending upon the plant species and the Agrobacterium delivery
system. A widely used approach is the leaf disc procedure which can
be performed with any tissue explant that provides a good source
for initiation of whole plant differentiation. Horsch et al. in
Plant Molecular Biology Manual A5, Kluwer Academic Publishers,
Dordrecht (1988) p. 1-9. A supplementary approach employs the
Agrobacterium delivery system in combination with vacuum
infiltration. The Agrobacterium system is especially viable in the
creation of transgenic dicotyledonous plants.
[0138] There are various methods of direct DNA transfer into plant
cells. In electroporation, the protoplasts are briefly exposed to a
strong electric field. In microinjection, the DNA is mechanically
injected directly into the cells using very small micropipettes. In
microparticle bombardment, the DNA is adsorbed on microprojectiles
such as magnesium sulfate crystals or tungsten particles, and the
microprojectiles are physically accelerated into cells or plant
tissues.
[0139] Following stable transformation plant propagation is
exercised. The most common method of plant propagation is by seed.
Regeneration by seed propagation, however, has the deficiency that
due to heterozygosity there is a lack of uniformity in the crop,
since seeds are produced by plants according to the genetic
variances governed by Mendelian rules. Basically, each seed is
genetically different and each will grow with its own specific
traits. Therefore, it is preferred that the transformed plant be
produced such that the regenerated plant has the identical traits
and characteristics of the parent transgenic plant. Therefore, it
is preferred that the transformed plant be regenerated by
micropropagation which provides a rapid, consistent reproduction of
the transformed plants.
[0140] Micropropagation is a process of growing new generation
plants from a single piece of tissue that has been excised from a
selected parent plant or cultivar. This process permits the mass
reproduction of plants having the preferred tissue expressing the
fusion protein. The new generation plants which are produced are
genetically identical to, and have all of the characteristics of,
the original plant. Micropropagation allows mass production of
quality plant material in a short period of time and offers a rapid
multiplication of selected cultivars in the preservation of the
characteristics of the original transgenic or transformed plant.
The advantages of cloning plants are the speed of plant
multiplication and the quality and uniformity of plants
produced.
[0141] Micropropagation is a multi-stage procedure that requires
alteration of culture medium or growth conditions between stages.
Thus, the micropropagation process involves four basic stages:
Stage one, initial tissue culturing; stage two, tissue culture
multiplication; stage three, differentiation and plant formation;
and stage four, greenhouse culturing and hardening. During stage
one, initial tissue culturing, the tissue culture is established
and certified contaminant-free. During stage two, the initial
tissue culture is multiplied until a sufficient number of tissue
samples are produced to meet production goals. During stage three,
the tissue samples grown in stage two are divided and grown into
individual plantlets. At stage four, the transformed plantlets are
transferred to a greenhouse for hardening where the plants'
tolerance to light is gradually increased so that it can be grown
in the natural environment.
[0142] Preferably, mature transformed plants generated as described
above are further selected for increase biomass, alcohol
production, vigor and/or yield.
[0143] Although stable transformation is presently preferred,
transient transformation of leaf cells, meristematic cells or the
whole plant is also envisaged by the present invention.
[0144] Transient transformation can be effected by any of the
direct DNA transfer methods described above or by viral infection
using modified plant viruses.
[0145] Viruses that have been shown to be useful for the
transformation of plant hosts include CaMV, TMV and BV.
Transformation of plants using plant viruses is described in U.S.
Pat. No. 4,855,237 (BGV), EP-A 67,553 (TMV), Japanese Published
Application No. 63-14693 (TMV), EPA 194,809 (BV), EPA 278,667 (BV);
and Gluzman, Y. et al., Communications in Molecular Biology: Viral
Vectors, Cold Spring Harbor Laboratory, New York, pp. 172-189
(1988). Pseudovirus particles for use in expressing foreign DNA in
many hosts, including plants, is described in WO 87/06261.
[0146] Preferably, the virus of the present invention is avirulent
and thus is incapable of causing severe symptoms such as reduced
growth rate, mosaic, ring spots, leaf roll, yellowing, streaking,
pox formation, tumor formation and pitting. A suitable avirulent
virus may be a naturally occurring avirulent virus or an
artificially attenuated virus. Virus attenuation may be effected by
using methods well known in the art including, but not limited to,
sub-lethal heating, chemical treatment or by directed mutagenesis
techniques such as described, for example, by Kurihara and Watanabe
(Molecular Plant Pathology 4:259-269, 2003), Gal-on et al. (1992),
Atreya et al. (1992) and Huet et al. (1994).
[0147] Suitable virus strains can be obtained from available
sources such as, for example, the American Type culture Collection
(ATCC) or by isolation from infected plants. Isolation of viruses
from infected plant tissues can be effected by techniques well
known in the art such as described, for example by Foster and
Tatlor, Eds. "Plant Virology Protocols From Virus Isolation to
Transgenic Resistance (Methods in Molecular Biology (Humana Pr),
Vol 81)", Humana Press, 1998. Briefly, tissues of an infected plant
believed to contain a high concentration of a suitable virus,
preferably young leaves and flower petals, are ground in a buffer
solution (e.g., phosphate buffer solution) to produce a virus
infected sap which can be used in subsequent inoculations.
[0148] Construction of plant RNA viruses for the introduction and
expression of non-viral exogenous polynucleotide sequences in
plants is demonstrated by the above references as well as by
Dawson, W. O. et al., Virology (1989) 172:285-292; Takamatsu et al.
EMBO J. (1987) 6:307-311; French et al. Science (1986)
231:1294-1297; and Takamatsu et al. FEBS Letters (1990)
269:73-76.
[0149] When the virus is a DNA virus, suitable modifications can be
made to the virus itself. Alternatively, the virus can first be
cloned into a bacterial plasmid for ease of constructing the
desired viral vector with the foreign DNA. The virus can then be
excised from the plasmid. If the virus is a DNA virus, a bacterial
origin of replication can be attached to the viral DNA, which is
then replicated by the bacteria. Transcription and translation of
this DNA will produce the coat protein which will encapsidate the
viral DNA. If the virus is an RNA virus, the virus is generally
cloned as a cDNA and inserted into a plasmid. The plasmid is then
used to make all of the constructions. The RNA virus is then
produced by transcribing the viral sequence of the plasmid and
translation of the viral genes to produce the coat protein(s) which
encapsidate the viral RNA.
[0150] Construction of plant RNA viruses for the introduction and
expression in plants of non-viral exogenous polynucleotide
sequences such as those included in the construct of the present
invention is demonstrated by the above references as well as in
U.S. Pat. No. 5,316,931.
[0151] In one embodiment, a plant viral polynucleotide is provided
in which the native coat protein coding sequence has been deleted
from a viral polynucleotide, a non-native plant viral coat protein
coding sequence and a non-native promoter, preferably the
subgenomic promoter of the non-native coat protein coding sequence,
capable of expression in the plant host, packaging of the
recombinant plant viral polynucleotide, and ensuring a systemic
infection of the host by the recombinant plant viral
polynucleotide, has been inserted. Alternatively, the coat protein
gene may be inactivated by insertion of the non-native
polynucleotide sequence within it, such that a protein is produced.
The recombinant plant viral polynucleotide may contain one or more
additional non-native subgenomic promoters. Each non-native
subgenomic promoter is capable of transcribing or expressing
adjacent genes or polynucleotide sequences in the plant host and
incapable of recombination with each other and with native
subgenomic promoters. Non-native (foreign) polynucleotide sequences
may be inserted adjacent the native plant viral subgenomic promoter
or the native and a non-native plant viral subgenomic promoters if
more than one polynucleotide sequence is included. The non-native
polynucleotide sequences are transcribed or expressed in the host
plant under control of the subgenomic promoter to produce the
desired products.
[0152] In a second embodiment, a recombinant plant viral
polynucleotide is provided as in the first embodiment except that
the native coat protein coding sequence is placed adjacent one of
the non-native coat protein subgenomic promoters instead of a
non-native coat protein coding sequence.
[0153] In a third embodiment, a recombinant plant viral
polynucleotide is provided in which the native coat protein gene is
adjacent its subgenomic promoter and one or more non-native
subgenomic promoters have been inserted into the viral
polynucleotide. The inserted non-native subgenomic promoters are
capable of transcribing or expressing adjacent genes in a plant
host and are incapable of recombination with each other and with
native subgenomic promoters. Non-native polynucleotide sequences
may be inserted adjacent the non-native subgenomic plant viral
promoters such that the sequences are transcribed or expressed in
the host plant under control of the subgenomic promoters to produce
the desired product.
[0154] In a fourth embodiment, a recombinant plant viral
polynucleotide is provided as in the third embodiment except that
the native coat protein coding sequence is replaced by a non-native
coat protein coding sequence.
[0155] The viral vectors are encapsidated by the coat proteins
encoded by the recombinant plant viral polynucleotide to produce a
recombinant plant virus. The recombinant plant viral polynucleotide
or recombinant plant virus is used to infect appropriate host
plants. The recombinant plant viral polynucleotide is capable of
replication in the host, systemic spread in the host, and
transcription or expression of foreign gene(s) (exogenous
polynucleotide) in the host to produce the desired protein.
[0156] Techniques for inoculation of viruses to plants may be found
in Foster and Taylor, eds. "Plant Virology Protocols: From Virus
Isolation to Transgenic Resistance (Methods in Molecular Biology
(Humana Pr), Vol 81)", Humana Press, 1998; Maramorosh and
Koprowski, eds. "Methods in Virology" 7 vols, Academic Press, New
York 1967-1984; Hill, S. A. "Methods in Plant Virology", Blackwell,
Oxford, 1984; Walkey, D. G. A. "Applied Plant Virology", Wiley, New
York, 1985; and Kado and Agrawa, eds. "Principles and Techniques in
Plant Virology", Van Nostrand-Reinhold, New York.
[0157] In addition to the above, the polynucleotide of the present
invention can also be introduced into a chloroplast genome thereby
enabling chloroplast expression.
[0158] A technique for introducing exogenous polynucleotide
sequences to the genome of the chloroplasts is known. This
technique involves the following procedures. First, plant cells are
chemically treated so as to reduce the number of chloroplasts per
cell to about one. Then, the exogenous polynucleotide is introduced
via particle bombardment into the cells with the aim of introducing
at least one exogenous polynucleotide molecule into the
chloroplasts. The exogenous polynucleotides selected such that it
is integratable into the chloroplast's genome via homologous
recombination which is readily effected by enzymes inherent to the
chloroplast. To this end, the exogenous polynucleotide includes, in
addition to a gene of interest, at least one polynucleotide stretch
which is derived from the chloroplast's genome. In addition, the
exogenous polynucleotide includes a selectable marker, which serves
by sequential selection procedures to ascertain that all or
substantially all of the copies of the chloroplast genomes
following such selection will include the exogenous polynucleotide.
Further details relating to this technique are found in U.S. Pat.
Nos. 4,945,050; and 5,693,507 which are incorporated herein by
reference. A polypeptide can thus be produced by the protein
expression system of the chloroplast and become integrated into the
chloroplast's inner membrane.
[0159] Since increase in biomass in plants can involve multiple
genes acting additively or in synergy (see, for example, in Quesda
et al., Plant Physiol. 130:951-063, 2002), the present invention
also envisages expressing a plurality of exogenous polynucleotides
in a single host plant to thereby achieve enhanced biomass
increases.
[0160] For example, the present invention contemplates
co-expression of exogenous UGPase (EC 2.7.7.9). According to one
embodiment the UGPase comprises an amino acid sequence at least 95%
homologous, and/or at least 90% identical to SEQ ID NO: 35 as
determined using the BlastP software of the National Center of
Biotechnology Information (NCBI) using default parameters. Thus,
for example the present invention contemplates expression of a
polypeptide as set forth by SEQ ID NO: 35 by expression of a
polynucleotide as set forth by SEQ ID NO: 36.
[0161] The nucleic acid sequence may be at least about 70%, at
least about 75%, at least about 80%, at least about 81%, at least
about 82%, at least about 83%, at least about 84%, at least about
85%, at least about 86%, at least about 87%, at least about 88%, at
least about 89%, at least about 90%, at least about 91%, at least
about 92%, at least about 93%, at least about 93%, at least about
94%, at least about 95%, at least about 96%, at least about 97%, at
least about 98%, at least about 99%, or more say 100% identical to
a nucleotide sequence selected from the sequence set forth by SEQ
ID NO: 36.
[0162] Nucleic acid sequences may encode polypeptide sequences
comprising an amino acid sequence at least about 70%, at least
about 75%, at least about 80%, at least about 81%, at least about
82%, at least about 83%, at least about 84%, at least about 85%, at
least about 86%, at least about 87%, at least about 88%, at least
about 89%, at least about 90%, at least about 91%, at least about
92%, at least about 93%, at least about 93%, at least about 94%, at
least about 95%, at least about 96%, at least about 97%, at least
about 98%, at least about 99%, or more say 100% homologous to SEQ
ID NO 35.
[0163] The present invention also contemplates co-expression of an
exogenous uridyl transferase UT, (EC 2.7.7.12). According to one
embodiment the UT comprises an amino acid sequence at least 85%
homologous, and/or at least 75% identical to SEQ ID NO: 37 as
determined using the BlastP software of the National Center of
Biotechnology Information (NCBI) using default parameters. Thus,
for example the present invention contemplates expression of a
polypeptide as set forth by SEQ ID NO: 37 by expression of a
polynucleotide as set forth by SEQ ID NO: 38.
[0164] The nucleic acid sequence may be at least about 70%, at
least about 75%, at least about 80%, at least about 81%, at least
about 82%, at least about 83%, at least about 84%, at least about
85%, at least about 86%, at least about 87%, at least about 88%, at
least about 89%, at least about 90%, at least about 91%, at least
about 92%, at least about 93%, at least about 93%, at least about
94%, at least about 95%, at least about 96%, at least about 97%, at
least about 98%, at least about 99%, or more say 100% identical to
a nucleotide sequence selected from the sequence set forth by SEQ
ID NO: 38.
[0165] Nucleic acid sequences may encode polypeptide sequences
comprising an amino acid sequence at least about 70%, at least
about 75%, at least about 80%, at least about 81%, at least about
82%, at least about 83%, at least about 84%, at least about 85%, at
least about 86%, at least about 87%, at least about 88%, at least
about 89%, at least about 90%, at least about 91%, at least about
92%, at least about 93%, at least about 93%, at least about 94%, at
least about 95%, at least about 96%, at least about 97%, at least
about 98%, at least about 99%, or more say 100% homologous to SEQ
ID NO 37.
[0166] Other polypeptides that may be co-expressed together with
the UGPPases of the present invention include epimerases.
[0167] Expressing a plurality of exogenous polynucleotides in a
single host plant can be effected by co-introducing multiple
nucleic acid constructs, each including a different exogenous
polynucleotide, into a single plant cell. The transformed cell can
than be regenerated into a mature plant using the methods described
hereinabove.
[0168] Alternatively, expressing a plurality of exogenous
polynucleotides in a single host plant can be effected by
co-introducing into a single plant-cell a single nucleic-acid
construct including a plurality of different exogenous
polynucleotides. Such a construct can be designed with a single
promoter sequence which can transcribe a polycistronic message
including all the different exogenous polynucleotide sequences. To
enable co-translation of the different polypeptides encoded by the
polycistronic message, the polynucleotide sequences can be
inter-linked via an internal ribosome entry site (IRES) sequence
which facilitates translation of polynucleotide sequences
positioned downstream of the IRES sequence. In this case, a
transcribed polycistronic RNA molecule encoding the different
polypeptides described above will be translated from both the
capped 5' end and the two internal IRES sequences of the
polycistronic RNA molecule to thereby produce in the cell all
different polypeptides. Alternatively, the construct can include
several promoter sequences each linked to a different exogenous
polynucleotide sequence.
[0169] The plant cell transformed with the construct including a
plurality of different exogenous polynucleotides, can be
regenerated into a mature plant, using the methods described
hereinabove.
[0170] Alternatively, expressing a plurality of exogenous
polynucleotides in a single host plant can be effected by
introducing different nucleic acid constructs, including different
exogenous polynucleotides, into a plurality of plants. The
regenerated transformed plants can then be cross-bred and resultant
progeny selected for superior biomass traits, using conventional
plant breeding techniques.
[0171] Hence, the present application provides methods of utilizing
novel genes to increase biomass in a wide range of economical
plants, safely and cost effectively.
[0172] As used herein the term "about" refers to .+-.10%.
[0173] Additional objects, advantages, and novel features of the
present invention will become apparent to one ordinarily skilled in
the art upon examination of the following examples, which are not
intended to be limiting. Additionally, each of the various
embodiments and aspects of the present invention as delineated
hereinabove and as claimed in the claims section below finds
experimental support in the following examples.
EXAMPLES
[0174] Reference is now made to the following examples, which
together with the above descriptions, illustrate the invention in a
non limiting fashion.
[0175] Generally, the nomenclature used herein and the laboratory
procedures utilized in the present invention include molecular,
biochemical, microbiological and recombinant DNA techniques. Such
techniques are thoroughly explained in the literature. See, for
example, "Molecular Cloning: A laboratory Manual" Sambrook et al.,
(1989); "Current Protocols in Molecular Biology" Volumes I-III
Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in
Molecular Biology", John Wiley and Sons, Baltimore, Md. (1989);
Perbal, "A Practical Guide to Molecular Cloning", John Wiley &
Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific
American Books, New York; Birren et al. (eds) "Genome Analysis: A
Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory
Press, New York (1998); methodologies as set forth in U.S. Pat.
Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057;
"Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E.,
ed. (1994); "Culture of Animal Cells--A Manual of Basic Technique"
by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; "Current
Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994);
Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition),
Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi
(eds), "Selected Methods in Cellular Immunology", W.H. Freeman and
Co., New York (1980); available immunoassays are extensively
described in the patent and scientific literature, see, for
example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;
3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;
3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and
5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984);
"Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds.
(1985); "Transcription and Translation" Hames, B. D., and Higgins
S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed.
(1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A
Practical Guide to Molecular Cloning" Perbal, B., (1984) and
"Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols:
A Guide To Methods And Applications", Academic Press, San Diego,
Calif. (1990); Marshak et al., "Strategies for Protein Purification
and Characterization--A Laboratory Course Manual" CSHL Press
(1996); all of which are incorporated by reference as if fully set
forth herein. Other general references are provided throughout this
document. The procedures therein are believed to be well known in
the art and are provided for the convenience of the reader. All the
information contained therein is incorporated herein by
reference.
[0176] General Materials and Methods
[0177] 1. Gene Cloning
[0178] UGGPase protein purification and peptide sequencing: Fresh
tissue of melon fruitlets (Cucumis melo, subsp. melo Group
Reticulatus, cv. Noy Yizre'el, 3 DAA, 3.5 g) was ground in liq N
and protein was extracted with 20 ml of buffer containing 50 mM
HEPES-NaOH (pH 7.5), 1 mM EDTA, 5 mM DTT, 1 mM PMSF, 0.1% PVP.
After centrifugation at 10 000 g for 30 min the supernatant was
filtered through 0.2 .mu.m cellulose acetate filter (Schleicher
& Schuell, Germany) and loaded on a MonoQ HR 5/5 (Pharmacia
Biotech AB, Uppsala, Sweden) column as described in Petreikov et
al. (2001). The column was equilibrated and the unbound protein
washed out with 20 mM HEPES-NaOH (pH 7.2), 2.5 mM DTT (buffer A),
and the bound protein was eluted with the 0-0.25 mM KCl gradient in
the same buffer. Protein of the fractions exhibiting UDP-Gal/Glu
PPase activity (described below) were concentrated by acetone
precipitation and subjected to SDS-PAGE (10%), using a Bio-Rad
Mini-Electrophoresis System according to the manufacturer's
instruction. 40 .mu.g and 10 .mu.g of protein were loaded for
Coomassie Brilliant Blue R-250 and immunoblotting, respectively.
Polyclonal UDP-Gal PPase antibodies, developed as described by
Feusi et al. [Feusi et al., (1999), Physiol Plant 106: 9-16] were
used for immunoblotting at a dilution of 1:1000. Following
immunoblotting, as described in Schaffer and Petreikov, (1997),
Plant physiol 113: 739-746, the UDP-Gal/Glu PPase band was
visualized using 5-bromo-4-chloro-3 indolyl phosphate/nitroblue
tetrazolium (Promega, Madison Wis., USA), according to the
manufacturer's instructions.
[0179] Peptide sequencing: The excised 68 kDa gel band stained with
Coomassie Blue and corresponding to the band of UGGPase as
determined by immunoblotting was subjected to MS/MS analysis
following protein digestion treatment with trypsin, further mass
spectrometry carried out with Otof2 (Micromass, England) using
nanospray attachment and data analysis (Otof Laboratory
Interdepartmental Equipment Unit, The Hebrew University Medical
School, Israel). Seven peptide sequences were obtained and a BLAST
analysis was carried out, identifying a homologous Arabidopsis gene
At5g52560 of unknown function. Using the Arabidopsis protein
sequence as a query, additional homologous genes were identified
from the various plant EST data bases available at TIGR
(www.tigr.org). The following ESTs were identified and used to
perform a CLUSTAL W homology alignment to identify conserved
sequences: wheat (TC251010), rice (TC277270), barley
(TC148351).
[0180] UGGPase cloning: The initial DNA fragment of the melon
UGGPase gene was cloned from young melon fruit cDNA using two
degenerate primers: UGGP-F1 5'GCN GGN YTN AAR TGG GT3' (SEQ ID NO:
1) and UGGP-R1 5'GGC CAN ACY TCN ACY TC3', (SEQ ID NO: 2) based on
the sequences AGLKWV (SEQ ID NO: 3) and EVEVWP (SEQ ID NO: 4). The
546 bp product was sequenced and cloning of the upstream region of
the gene was carried out using the upstream degenerate primer
UGGP-F3 5'TCN AGY TAY CCN GGN GG3' (SEQ ID NO: 5), for the SSYPGG
(SEQ ID NO: 6), N-terminal sequence together with degenerate primer
UGGP-R1 (SEQ ID NO: 2). The UGGPase full length sequence was cloned
from a young melon fruit EST library (see below) using: (1) UGGPase
internal primers: UGGP-R5' 5'CCCACCAGCAACAAGAACAAA3' (SEQ ID NO: 7)
and UGGP-F3' 5'CTTCAACCCGATTGGAATGTA3' (SEQ ID NO: 8) and (2)
primers of T7 and T3 promoter sequences from the multiple cloning
site region of the pBK-CMV phagemid vector.
[0181] Young fruit EST library: Total RNA was isolated from 10 gr
fresh weight from a mixture of `Noy Yizre'el` fruits, collected at
a 0, 1, 3, 12, and 25 DPA, using a modification of the method of La
Claire and Herrin (1997), Plant Mol. Bio. Rep. 15: 263-272. Poly
(A).sup.+ mRNA was purified from 1 mg of total RNA by use of
Oligotex.TM. mRNA purification kit according to manufacturer's
recommendations (Qiagen, Hilden, Germany). 5 .mu.g of poly
(A).sup.+ mRNA was used for preparation of the library. The EST
library was constructed using ZAP cDNA Synthesis Kit and ZAP
Express.TM. cDNA Gigapack.TM. III Gold Cloning Kit according to
manufacturer's recommendations (Stratagene, La Jolla, Calif.,
U.S.A.). Phage clones were mass-excised to pBK-CMV phagemid vector
following the manufacturer's instructions (Stratagene, La Jolla,
Calif., U.S.A.).
[0182] UGPase cloning: Melon UGPase was cloned from young melon
cDNA using four degenerate primers: UGP-F1 5'ACN ATG GGN TGY CAN
GG3' (SEQ ID NO: 9); UGP-F2 5'GAY GGN TGG TAY CCN CC3' (SEQ ID NO:
10); UGP-R1, 5'CCN CCY TTN ACR TCN GC3' (SEQ ID NO: 11) and UGP-R2
5'CCR TCN ACY TCY TTN GG3' (SEQ ID NO: 12). The degenerate primers
were constructed based on consensus amino acid sequences (TMGCTG
(SEQ ID NO: 13), DGWYPP (SEQ ID NO: 14), ADVKGG (SEQ ID NO: 15),
PKEVDG (SEQ ID NO: 16), respectively) identified by multiple
alignments of potato (AAB71613), Arabidopsis (AKK64100), banana
(AAF17422), rice (BAB69069), barley (CAA62689) and Japanese pear
(BAA25917) published full length sequences. The initial 705 bp PCR
fragment of the melon UGPase gene was cloned and sequenced using
the T/A cloning vector pGEM-Teasy (Promega, Madison, Wis., USA).
Melon UGPase full length sequence was cloned from a young melon
(cv. Noy Yizre'el) cDNA library constructed in a yeast shuttle
vector, pFL61 (Minet et al., 1992). The internal primers UGP-F202
5' ACATTCAACCAGAGCCAATATC3' (SEQ ID NO: 17) and UGP-R603
5'CACCCACAAATTGTTAGTGTTG3' (SEQ ID NO: 18) together with the pFL61
flanking region primers pFL-F and pFL-R were used to clone the
UGPase 5' and 3' regions and assemble the full gene sequence.
[0183] Uridyltransferase cloning: The cloning of melon Gal-1-P
uridyltranferase (UT) gene from melon was also carried out based on
conserved amino acid sequences. However, when this cloning work was
started there were only a few partial plant ESTs in the databases,
in addition to human, bacterial and fungal UTs and an Arabidopsis
UT. Based on the CLUSTAL W homology alignment from plant UT
sequences of tomato (TC103202), potato (TC41313), wheat (TC46973)
and Arabidopsis (NM.sub.--121825) conserved sequences of
E(H/Q)(E/Q)CAPE and QVFKN(Q/H)GA were selected for the preparation
of degenerate primers. The first 320 bp of the melon Gal-1-P
uridyltranferase (UT) gene was cloned by the degenerate primers:
UT-F7 5'GAG CAN SAG TGY GCN CCN GAG3' (SEQ ID NO: 19) and UT-R7
5'GCN CCN TGG TTY TTG AAN ACC TG3' (SEQ ID NO: 20). Full sequence
of the melon-UT was completed by direct sequencing of the BAC clone
121K16 selected from the CUGI (www.cugi.edu) melon BAC library MR-1
EcoR1 filters using the UT 320 bp fragment as a probe. Cloning was
done from the BAC due to the very low abundance of UT mRNA in melon
fruit. The primers: UT-R 5' ACATCCTCGGGGGTCAAATCAGA3' (SEQ ID NO:
21) and UT-F3' 5'CAGGCTTCGGATTCAGACTTAG3' (SEQ ID NO: 22) were used
to sequence the melon UT 5' end and 3' end from the BAC DNA,
respectively.
[0184] 2. Expression of UGPase, UGGPase and UT mRNA in E. coli.
[0185] Full length open reading frames were cloned in the bacterial
expression vector pET-28a (Novagen, EMD Biosciences, San Diego,
Calif., USA) using the following restriction sites: NdeI site for
5' end of the three ORFs; XhoI site for 3' end of the UGPase and
UGGPase; and BamHI site for 3' end of UT.
[0186] Expression plasmids containing UGPase, UGGPase or UT mRNA
were transformed into E. coli BL21 (DE3) LysE cells (Dubendorff and
Studier, 1991). Bacterial colonies were grown in a 50-ml flask
containing 10 ml of LB medium to an OD.sub.600 of 0.5, induced for
expression with 0.4 mM of IPTG, (control bacterial extracts were
prepared from non-induced (-IPTG) cultures) and harvested after 6
hours or overnight by centrifugation at 4000 g for 10 min. Cells
were lysed by re-suspension in 2 ml extraction buffer (20 mM
phosphate buffer pH 8, 1 mM EDTA, 500 mM NaCl, 0.1% Triton x100,
2.5 mm DTT and 1 mg/ml lysozyme) for 1 h at 4.degree. C., and
mechanically broken by freezing and thawing three times. The
viscous bacterial lysate was sheared using a 21 gauge needle and
crude soluble protein extract was obtained after centrifugation at
15,000 g for 30 min at 4.degree. C. and collection of the soluble
fraction. The bacterial crude proteins extracts were used to assay
enzyme activities.
[0187] 3. RNA Extraction Northern-Blot Analysis.
[0188] Total RNA was isolated from 10 gr fresh weight of melon
flesh using a modification of the method of La Claire and Herrin
(1997), Plant Mol. Bio. Rep. 15: 263-272. Each 10 gr sample was
pooled from 3 melon fruit of the same developmental stage. For
northern-blot analysis, 20 .mu.g of total RNA from each
developmental stage was separated on a denaturing 1% agarose-gel
using Mops buffer. Expression of UGPase and UGGPase mRNAs were
analyzed by RNA gel blotting using specific probes for UGPase (422
bp) and UGGPas (710 bp). Detection of UGPase and UGGPase mRNAs was
analyzed using a Phospholmager (Molecular Dynamics, Sunnyvale,
Calif.). To indicate the amount of RNA loaded in each well the
nylon membrane was stained for 5 min with 5% methylene-blue.
[0189] 4. Quantitative Real-Time PCR.
[0190] cDNA was synthesized from 1 .mu.g RNA (DNase-treated) using
the Reverse-iT.TM. 1.sup.st Strand Synthesis Kit (ABgene, Surrey,
UK), according to manufacturer's instructions. 1 .mu.l cDNA product
was used as template for real-time PCR reaction based on Eurogentec
gPCR.TM. core kit and SYBR.sup.R Green I as a fluorescent
substance. The specific primers used for the UGGPase and UT genes
were: UGGP-QF 5' AACCCGATTGGAATGTATGAT3' (SEQ ID NO: 23); UGGP-QR
5'CCGAAGTAGCACTGTGATAAG3' (SEQ ID NO: 24), and UT-QF 3'
TCCTGCTCTCAGTAGGGATAAGG5 (SEQ ID NO: 25)'; UT-QR
5'ACATCCTCGGGGGTCAAATCAGA3' (SEQ ID NO: 26). The melon Actin cDNA
(AY859055) was quantified with the following primers: forward,
5'GATTCCGTGCCCAGAAGTT3' (SEQ ID NO: 27) and reverse,
5'TTCCTTGCTCATCCTGTCTG3' (SEQ ID NO: 28) and used for normalizing
the expression data. The real-time PCR reaction was initiated by
heat activation of 10 min at 95.degree. C. and continued for 40
cycles of 15 s at 95.degree. C., 30 s at 60.degree. C., and 30 s at
72.degree. C., using the GeneAmp 5700 Sequence Detection System (PE
Biosystems). Each specific amplicon: 167 bp for UGGPase, 159 bp for
UT and 187 bp for the melon Actin genes had only one dissociation
peak (not shown) and linear calibration curves (for all genes,
R.sup.2=0.96-0.99). The specific gene expression was calculated
relative to the actin mRNA level in each sample according to the
equation 2.sup.-(Ct sample-Ct actin), where Ct is the threshold
cycle of the specific gene and actin.
[0191] 5. Enzyme Extraction.
[0192] Assays of native fruit enzyme activities were carried out on
the crude extracts as described in the enzyme purification section
above. When enzyme fractions were separated by ion exchange
chromatography, conditions were as described above with the
exception of the separated tomato fruit extract in which the
extraction buffer and elution buffer consisted of BisT Propane (pH
9.0) in an attempt to bind the UGPase enzyme to the MonoQ
column.
[0193] Phenyl Sepharose Hydrophobic Interaction chromatography (Hi
Trap HIC, 1 ml, Pharmacia Biotech) was also used for the separation
of the melon UGGPase and UGPase enzymes. The extraction mixture
consisted of 50 mM Phosphate (pH 7.0), 2 mM EDTA, 5 mM MgCl.sub.2,
0.8 mM gal, 5 mM DTT, 1 mM PMSF. The supernatant after
centrifugation at 10,000 g for 30 min was adjusted to 1M ammonium
sulfate, incubated in ice for 20 min, centrifuged, filtered and
applied in the column. The unbound protein washed out with 50 mM
Phosphate (pH 7.0), 1 M ammonium sulfate and the bound protein was
eluted with the 1-0 M ammonium sulfate gradient in the phosphate
buffer.
[0194] The bacterial-expressed enzymes were extracted as described
above. For ion exchange chromatographic separation the enzyme
extracts were diluted in 25 mM HEPES-NaOH (pH 7.5), 1 mM EDTA, 5 mM
MgCl.sub.2, 0.5 mM DTT and separated by MonoQ anion-exchange
chromatography under conditions identical to the melon fruit enzyme
extract conditions described above.
[0195] 6. Enzyme Assays.
[0196] Nucleotide-sugar synthesis direction: In the synthesis
direction of UDP-sugars enzyme activities were assayed as described
in detail by Gao et al. [Gao et al, (1999), Physiol Plant 106: 1-8;
Gao et al, (1999), Plant Physiol 119: 979-988] using Glc-1-P or
Gal-1-P as substrates. The reaction mixture, in a total volume of
0.2 ml, contained 25 mM HEPES-NaOH pH 7.5, 1 mM EDTA, 5 mM
MgCl.sub.2, 0.5 mM DTT, 10 mM Gal-1-P or Glc-1-P and 2.5 mM UTP.
The reaction was initiated by adding 20 .mu.l enzyme preparation at
30.degree. C. and terminated after 3 min by 2 min boiling. After
cooling to room temperature, 1 ml 50 mM Tricine buffer pH 8.7
containing 0.5 mM NAD, 0.01 unit of UDP-Glc dehydrogenase (Sigma)
and 0.02 unit of UDP-Glc-4' epimerase (Sigma) was added and the
mixture was incubated at 30.degree. C. for 1 hr prior to measuring
340 nm. Enzyme activity was expressed as .mu.mol UDP-Gal produced
per min at 30.degree. C.
[0197] For the determination of the kinetic parameters of the
partially purified enzymes the substrates Glc-1-P and Gal-1-P were
used in concentrations from 0 to 5 mM. The amount of UDP-sugars
produced was quantified from standard curves of 0-75 nmol UDP-Glc
and UDP-Gal in 0.5 ml of reaction mixture under the same assay
conditions and activity was expressed as the production of .mu.mol
UDP-Glc or UDP-Gal per min at 30.degree. C.
[0198] Pyrophosphorolytic direction: In the pyrophosphorolytic
direction the sugar-phosphate production was measured according to
Smart and Pharr (1981) Planta 153: 370-375, with modifications as
follows. The reaction buffer contained 25 mM HEPES-NaOH (pH 7.5), 1
mM EDTA, 5 mM MgCl.sub.2, 0.5 mM DTT with addition of 1 mM of
either UDP-Glc or UDP-Gal and 10 .mu.l of partially purified enzyme
sample in a 100 .mu.l reaction mixture. The reaction was initiated
by 1 mM PPi and stopped after 3 min by boiling for 2 min and the
mixture was cooled on ice. For the measurement of the respective
hexose-1-P product a single mixture was added: 400 .mu.l of 50 mM
HEPES-NaOH pH (7.8) containing 5 mM MgCl.sub.2, 4 mM UDPG, 0.02 U
Gal-1-P Uridyltransferase (Sigma), 1 mM NAD, 10 .mu.M g1-1,6 bis P,
2 U Phosphoglucomutase (PGM) and 1 U Glc-6-P dehydrogenase (G6PDH,
from Leuconostoc). After 40 min incubation at 30.degree. C.,
absorbance of NADH product was recorded at 340 nm.
[0199] For the determination of kinetic parameters the substrates
UDP-Glc or UDP-Gal were used in concentrations from 0 to 1 mM. The
amount of hexose-P produced was quantified from a standard curve of
0-100 nmol Glc-1-P/Gal-1-P in 0.5 ml of reaction mixture under the
same assay condition and expressed as .mu.mol Glc-1-P/Gal-1-P per
min at 30.degree. C.
[0200] For screening enzyme activities in the HPLC fractions during
purification two separate assays were used. For determining
activity with the Glc moiety a coupled continuous assay was used in
the pyrophosphorolytic direction and Glc-1-P formation was
monitored as in Schaffer and Petreikov (1997), Plant physiol 113:
739-746. In brief, the PPi-dependent production of Glc-1-P from
UDP-Glc was measured in a linked assay containing NADH, PGM and
G6PDH. For the determination of fractions active with the Gal
moiety we used the Gal-1-P specific assay described above in the
nucleotide-sugar synthesis direction.
[0201] Gal-1-Phospate uridyltransferase (UT): Two separate methods
were used to measure UT activity in light of the near absence of
activity in melon fruit. A continuous coupled enzyme assay modified
from Elsevier and Fridovich-Keil (1996), Biol Chem 271:
32002-32007, was carried out in a 0.5 ml reaction mixture
containing 50 mM HEPES-NaOH (pH 7.8), 5 mM MgCl.sub.2, 0.5 mM DTT,
10 mM Gal-1-P, 1 mM NAD, 10 .mu.M g1-1,6 bis P, 1 U G6PDH (from
Leuconostoc), 2 U PGM, and enzyme sample. The reaction was
initiated by 4 mM UDP-Glc and monitored for 10 min at 37.degree. C.
The amount of Glc-1-P produced was expressed as the amount of
enzyme necessary to produce 1 .mu.mol Glc-1-P per min at 37.degree.
C. Alternatively, a two-step end point assay, modified from Main et
al. (1983), Physiol Plant 59: 387-392, was used for determining UT
activity. The reaction buffer contained 25 mM HEPES-NaOH (pH 7.5),
1 mM EDTA, 5 mM MgCl.sub.2, 0.5 mM DTT with addition of 10 mM
Gal-1-P and 10 .mu.l of partially purified enzyme sample in a 100
.mu.l reaction mixture. The reaction was initiated by 4 mM UDP-Glc
and stopped after 10 min by boiling for 2 min and the mixture was
cooled on ice. For the measurement of the Glc-1-P product 400 .mu.l
consisting of 50 mM HEPES-NaOH pH (7.8) 5 mM MgCl.sub.2, 1 mM NAD,
10 .mu.M g1-1,6 bis P, 2 U PGM and 1 U G6PDH. After 40 min
incubation at 30.degree. C., absorbance of NADH product was
recorded at 340 nm.
[0202] 7. Protein Estimation
[0203] The Bio-Rad protein assay and BSA as a standard were used to
estimate the protein concentration according to the method of
Bradford et al. (1976), Anal Biochem 72: 248-254
Example 1
Purification and Peptide Sequencing and Cloning of UDP-Gal/Glc
Pyrophosphorylase, UGPase and UT
[0204] Results
[0205] Antibodies prepared against the purified protein of Feusi et
al [Feusi et al., 1999, Physiol Plant 106: 9-16] were used to
identify a partially purified protein extract from young melon
fruit (FIGS. 1A-C). The corresponding 68 kD band from the SDS-PAGE
gel was excised and the protein sequenced after partial peptide
hydrolysis.
[0206] Based on seven peptide sequences obtained (SEQ ID NOs:
39-45), a BLAST analysis was performed, identifying At5g52560 as a
homologous gene (79%) included in Pfam01704 and containing a UGPase
motif (FIG. 2A). Based on the homology with the Arabidopsis
homologue and additional plant homologues reported in the TIGR EST
databases, degenerate primers were synthesized and a 546 bp
amplified product was sequenced. The upstream and downstream
portions of the gene were sequenced from a young melon fruit cDNA
library in pBK-CMV phagemid vector. FIG. 2A shows the deduced
sequence of the protein and its homologies to similar plant
enzymes, as well as the seven peptide sequences obtained. The
calculated MW of the enzyme is 67,787 consistent with the band in
FIGS. 1B-C. This enzyme is referred to as UDP-Gal/Glc
pyrophosphorylase (UGGPase, deposited in gene bank as
DQ399739).
[0207] The genes for melon fruit UGPase and UT were cloned by PCR,
based on homologous and conserved sequences of other plant genes in
the database (as indicated in FIGS. 2B-C). Full length sequences
were obtained from the young fruit cDNA library and melon BAC
library for UGPase and UT, respectively. The UGPase gene encodes
for a protein of 52 kDa and the UT gene encodes for a protein of 38
kD, consistent with the MW of enzymes in these two families.
Example 2
Functional Expression and Characterization of the Gene Products
[0208] Results
[0209] FIGS. 3A-C show the heterologously expressed proteins of
UGGPase, UGPase, and UT in E. coli extracts. The expressed UGGPase,
UGPase, enzymes were active and were partially purified by ion
exchange chromatography and characterized with regard to substrate
specificity and affinity. The UT enzyme was sequestered in
inclusion bodies. Mass spectrometry analysis of the differentially
expressed band in FIG. 3C indicated that it is indeed UT (results
not shown).
[0210] The novel UGGPase can utilize both Glc-1-P and Gal-1-P in
the synthesis of the respective nucleotide sugars and also can
utilize either UDP-Glc or UDP-Gal in the reverse direction.
Substrate affinity is higher for the Glc moiety in both directions
but V.sub.max is higher for the Gal moiety in each direction as
illustrated in Table 1, herein below.
TABLE-US-00001 TABLE 1 pyrophosphorolysis UDP sugar synthesis
UDP-Gal UDP-Glc Gal-1-P Glc-1-P Enzyme Km Vmax Km Vmax Km Vmax Km
Vmax UGGPase 0.44 625 0.14 238 0.43 714 0.27 222 UGPase 0.26 714
0.11 277 ND ND 0.24 238 Km (mM) and Vmax (.mu.mol mg
protein.sup.-1min.sup.-1) of heterologously expressed and partially
purified melon UGGPase and UGPase. ND, Not detected.
[0211] The heterologously expressed melon UGPase is specific for
the Glc moiety in the direction of nucleotide sugar synthesis
(using Glc-1-P as substrate) and shows no observable activity with
Gal-1-P. However, in the reverse direction, the UGPase did show
activity with UDP-Gal, as well as with UDP-Glc. Affinity of the
UGPase for the UDP-Gal is slightly lower than for UDP-Glc; however,
the V.sub.max is significantly higher
[0212] In light of the surprising result that the melon UGPase is
active with UDP-Gal, the characteristics of the purified melon
UGPase were compared with those from a non-cucurbit plant, young
tomato fruit, in order to determine whether the ability to
metabolize UDP-Gal is unique to the melon UGPase. Surprisingly, it
was observed that a partially purified tomato fruit UGPase did, in
fact, metabolize UDP-Gal in addition to UDP-Glc as illustrated in
Table 2 herein below.
TABLE-US-00002 TABLE 2 Comparison of substrate specificity of melon
and tomato UGPase and UGGPase. Control non-induced transformed E.
coli extracts (-IPTG) showed ca 5% of the activity with either
UDP-Gal and UDP-Glc, as compared to the induced (+IPTG) E. coli
extracts. Enzyme activity, .mu.mol mg protein.sup.-1 min.sup.-1
melon melon tomato UGPase UGGPase UGPase Substrates E. coli E. coli
native UDP-Gal + PPi 460 410 12 UDP-Glc + PPi 245 233 38 Gal-1-P +
UTP ND 678 ND Glc-1-P + UTP 187 223 8 ND, Not detected.
[0213] The tomato enzyme fraction did not show any UDP-Glc-4'
epimerase activity (not shown), indicating that the activity
measured with UDP-Gal was not an artifact due to UDP-Gal to UDP-Glc
conversion. In the reverse direction the tomato UGPase was specific
for Glc-1-P and did not metabolize Gal-1-P, similar to the melon
UGPase and indicating that the partially purified fraction did not
contain a UGGPase.
Example 3
Gal Metabolism Gene Expression and Enzyme Activities in Young
Fruit
[0214] Results
[0215] In order to determine the potential relative contribution of
the three enzymes in Gal-1-P flux in developing melon fruit, crude
extracts were assayed from immature and developing ovaries and
compared to the relative quantitative expression of their
respective genes. The enzyme activities of the UGPase, UGGPase and
UT in developing ovaries are presented in Table 3 herein below.
TABLE-US-00003 TABLE 3 Activity of UGGPase, UGPase and UT in crude
extracts from young melon ovaries Activity Assay for (.mu.mol
product mg Substrate enzyme protein.sup.-1 min.sup.-1) Gal-1-P +
UTP UGGPase 8 UDP-Glc + PPi UGPase + 14 UGGPase UDP-Glc + Gal-1-P
UT 0.007
[0216] The assay of UGGPase was carried out using Gal-1-P as
substrate so that the assay was specific for this enzyme. However,
since both UGPase and UGGPase are active with the Glc moiety in
either direction the assay does not distinguish between the two
enzymes. The two activities were therefore separated using
hydrophobic interaction chromatography and the results show that
the two enzymes are of approximate equal activity (FIG. 4). The
results indicate that Gal-1-P metabolism is carried out
preferentially by the UGGPase enzyme. Although both the UGGPase as
well as the Glc-1-P specific UGPase are active in the developing
fruit, the latter is inactive on the galactokinase reaction product
Gal-1-P, as described above. UT activity is barely observed in the
developing ovaries (Table 3).
[0217] The gene expression patterns of the three genes paralleled
the enzyme activity in crude extracts of developing melon ovaries
and fruit. Northern blots showed expression of both UGGPase and
UGPase throughout fruit development while UT expression was not
observed at the level of detection of Northern blots (FIG. 5A).
Quantitative RT-PCR was performed on mRNA of melon ovaries and
developing fruit and UT expression was very low, compared to
UGGPase (FIG. 5B).
Example 4
Flux of Gal-1-P
[0218] Results
[0219] In order to prove that both pyrophosphorylase reactions in
the Gal-1-P to Glc-1-P flux can be carried out in consort by
UGGPase in the absence of UGPase, Glc-1-P production was measured
from the substrates Gal-1-P and UTP in the presence of only UGGPase
and epimerase. PPi was also not added in order to test whether the
second pyrophosphorylase reaction which is dependent on PPi can
take place dependent on the production of PPi in the initial
reaction. The partially purified native melon UGGPase (Table 4,
herein below), as well as the heterologously expressed melon
UGGPase (Table 5, herein below), were each used in conjunction with
a purified epimerase to make certain that UGPase activity was not
present. The Glc-1-P product was continuously removed by the linked
PGM and G6PDH reactions in an enzyme-linked assay. The results of
these experiments (as set forth in Tables 4 and 5, herein below)
show that the UGGPase alone can carry out both the Gal-1-P
conversion to UDP-Gal and the subsequent reverse reaction of
UDP-Glc to Glc-1-P. Most significantly, the synthesis of Glc-1-P
from UDP-Glc took place without the external addition of PPi,
indicating that the PPi produced in the
Gal-1-P+UTP.fwdarw.UDP-Gal+PPi reaction was cycled into the reverse
reaction following the epimerase step.
TABLE-US-00004 TABLE 4 Dependence of Glc-1-P production from
Gal-1-P and UTP on the addition of partially purified melon fruit
UGGPase and purified epimerase. Each reaction was carried out with
ca 13 .mu.g protein from fraction 18 of FIG. 1a in a 1 ml reaction
mix. Glc-1-P produced Enzyme in reaction (.mu.mol Glc-1-P mg
Substrate UGGPase epimerase protein.sup.-1 min.sup.-1) Gal-1-P, UTP
+ + 1.8 Gal-1-P, UTP + - ND Gal-1-P, UTP - - ND ND, no activity
detected
TABLE-US-00005 TABLE 5 Glc-1-P production from Gal-1-P and UTP with
crude extracts of E. coli expressed protein of either melon UGGPase
or melon UGPase, together with purified epimerase. The E. coli
extract (-IPTG) did not express the UGGPase protein and served as
blank reaction. Each reaction was carried out with ca 10 .mu.g
protein from the crude E. coli extractions shown in FIG. 3 in a 1
ml reaction mix. Glc-1-P produced (.mu.mol Glc-1-P mg Substrate E.
coli extract protein.sup.-1 min.sup.-1) Gal-1-P, UTP UGGPase, -IPTG
ND Gal-1-P, UTP UGGPase, +IPTG 14.5 Gal-1-P, UTP UGPase, +IPTG ND
ND, no activity detected.
Example 6
Tobacco Transformation with UGGPPase
[0220] Materials and Methods
[0221] Cloning of UGGPPase and plasmid construction for tobaco
transformation: UGGPPase was cloned from fresh melon fruitlets as
described above, and inserted into the pGA643 binary vector
(Genbank AY804024) with the cauliflower mosaic virus 35S
constitutive promoter (Benfey and Chua, 1990 Benfey, P. N. and
Chua, N.-H. (1990) The cauliflower mosaic virus 35S promoter:
combinatorial regulation of transcription in plants. Science, 250,
959-966). This plasmid also contained the nptII gene under the
control of Nopalin synthase (nos) promoter and terminator.
[0222] Plant Transformation and Maintenance
[0223] Nicotiana tabacum cv. Xanthi NN (tobacco) was transformed
using Agrobacterium tumefaciens strain EHA105 (Hood et al., 1993
Transgenic Res. 2, 208-218) employing a standard leaf disc
inoculation method. Binary plasmids were inserted into EHA105 via
electroporation and plated on LB-Agar medium with 50 mg/l
kanamycin. Tobacco Leaf discs were cut and plated abaxially on
Petri-dish containing D.sub.0 medium (Full MS (Murashige and
Skoog)+3% sucrose supplemented with 0.1 mg/ml of
.alpha.-naphthalene acetic acid (NAA) and 1 mg/ml
6-benzylaminopurine (BA), and solidified with 0.8% (w/v) plant-agar
(Duchefa)). After 24 hours of pre-cultivation, the leaf explants
were floated with logarithmic culture (OD.about.0.3) of the
transformant EHA105 supplemented with Acetosyringone (100 .mu.M
final concentration) and incubated for 1 hour at room temperature.
After 1 hour, the remaining bacterial suspension was pumped out and
the leaf discs were co-cultivated for 48 hours in the dark. The
explants were then transferred to selective regeneration medium
D.sub.1 (MS minerals with 400 mg/L carbenicillin, 70 mg/L
kanamycin, 0.1 mg/L NAA and 1 mg/L BA). Regenerated explants were
transferred to fresh medium biweekly. Green shoots, 1-3 cm tall,
were separated from calli and transferred to Rooting medium
containing full MS minirals, 200 mg/L carbenicillin, 75 mg/L
kanamycin and 1 mg/L Indole butyric acid (IBA). Rooted plants were
transplanted to peat cookies (Jiffy 7) for hardening and then grown
in 4 liter pots in the greenhouse.
[0224] Plants were confirmed as transgenic by PCR screening of
genomic DNA employing the nptII specific primers: NPT-F 5'
CACGCAGGTTCTCCGGCCGC 3' (SEQ ID NO: 29) and NPT-R 5'
TGCGCTGCGAATCGGGAGCG 3' (SEQ ID NO: 30) and gene-specific
oligonucleotides: GalPP-F 5' CAGCAATAGACTGGCAGGTGA 3' (SEQ ID NO:
31) and GalPP-R 5' CCAATCGGGTTGAAGACTTGA 3' (SEQ ID NO: 32).
Genomic DNA was isolated using the DNeasy Plant Mini Kit (Qiagen,
Mississauga, Ontario).
[0225] Plant growth: Primary transformed plants and control lines
(T.sub.0) were grown to maturity and self-fed to generate T.sub.1
lines of all the single transformants and the associated controls.
The pods were collected, and the seeds removed and sterilized by
washing for 2 min in a 10% bleach solution, followed by a 1-min
rinse in sterile water. Seeds were germinated on solid
half-strength MS medium with % sucrose and kanamycin (50 mg/L). The
surviving seedlings were then PCR screened using the aforementioned
primer sets. Seedlings were grown in GA-7 vessels prior to transfer
into 7.5-L pots containing a 50% peat-25% fine bark-25% pumice soil
mixture in the glasshouse, and covered with 16-oz clear plastic
cups for 1 week to aid in acclimation. Each line, transgenic and
control, was represented by 12 individual plants (each from an
individually selected seed).
[0226] Transcription levels: Semi quantitative RT-PCR was used to
determine the transcript level of each transgene. Leaf sections
weighing approximately 100 mg were ground in liquid nitrogen, and
RNA was extracted using EZ-RNA total RNA extraction kit reagent
(Biological Industries, Bet Haemek, Israel), according to the
manufacturer's instructions. Following extraction, 20 .mu.g of
total RNA was treated with 2 unit of DNase I (Fermentas) according
to the manufacturer's instructions. The reaction was incubated at
37.degree. C. for 30 min and then heat inactivated at 80.degree. C.
for 10 min.
[0227] Equal quantities of total RNA (2 .mu.g) were employed for
the synthesis of cDNA using RevertAid Hminus M-Mulv Reverse
Transcriptase (Fermentas) and oligo dT.sub.12-18 primer, and random
hexamers, according to the manufacturer's instructions. PCR was
carried out at Tm temperature of 62.degree. C. using 1 .mu.l of the
first-strand cDNA product of the above reaction as a templates and
UGGPase specific primers GalPP-F and GalPP-R (see above). Reactions
were run for both 25 and 35 cycles so that the results can be
interpreted in semi-quantitative manner. PCR reaction products were
run on 1% agarose gels, stained and photographed. The results of 25
cycles show that the expression levels of the UGGPPase gene varied
between the independent transformants (FIG. 8). Highest expression
was observed in UGGP4, 10, 11 and 23. UGGP19 and UGGP21 were
non-transformed individuals.
[0228] Enzyme activity of transgenic tobacco plants: Leaf samples
(approximately 400 mg fresh weight) were ground in liquid N and
protein was extracted with 1 ml of buffer containing 50 mM
HEPES-NaOH (pH 7.5), 1 mM EDTA, 5 mM DTT, 1 mM PMSF, 2% PVPP. After
centrifugation at 10 000g for 30 min the supernatant was used as
the crude enzyme extract. UGGPPase was assayed in the
nucleotide-sugar synthesis direction using gal-1-P as substrate, as
described above. In brief, the reaction mixture, in a total volume
of 0.1 ml, contained 25 mM HEPES-NaOH pH 7.5, 1 mM EDTA, 5 mM
MgCl.sub.2, 0.5 mM DTT, 10 mM Gal-1-P and 2.5 mM UTP. The reaction
was initiated by adding 10 .mu.l enzyme preparation at 30.degree.
C. and terminated after 3 min by 2 min boiling. After cooling to
room temperature, 0.4 ml 50 mM Tricine buffer pH 8.7 containing 0.5
mM NAD, 0.01 unit of UDP-Glc dehydrogenase (Sigma) and 0.02 unit of
UDP-Glc-4' epimerase (Sigma) was added and the mixture was
incubated at 30.degree. C. for 1 hr prior to measuring 340 nm.
Enzyme activity was expressed as .mu.mol UDP-Gal produced per min
at 30.degree. C.
[0229] Plant growth: The glasshouse plants were harvested at the
onset of flowering, as indicated by the formation of flower buds.
The plant height, from base to tip of the highest bud, was measured
prior to harvest. The developmental stages of tissues were
standardized by employing a plastichron index (PI) (PI=0 was
defined as the first leaf greater than 5 cm in length; PI=1 was the
leaf immediately below PI=0). A portion of the stem from each plant
spanning PI=5 to PI=15 was excised and immediately weighed for
total stem fresh weight measurements and leaf biomass. This same
section was then dried at 105.degree. C. for 48 h for dry weight
determination, and retained for further analysis. The internode
distance represents the average length between each internode
spanning PI=5 to PI=15. The lower section of the stem (below PI=15)
was dried at room temperature for fibre quality analysis. Data is
analyzed for growth rate on fresh and dry weight bases.
[0230] Soluble carbohydrate and starch analysis: Soluble
carbohydrates (glucose, fructose and sucrose) are extracted from
plant material (leaf, stem, roots) using five successive
extractions in hot (68.degree. C.) ethanol: H.sub.2O (80:20). The
ethanol is evaporated and the dried residue is suspended in double
distilled H.sub.2O, centrifuged to remove debris and filtered
through a 45 micron filter. Sugars are separated
chromatographically by HPLC (Shimadzu LC10AT) in a Bio-Rad Fast
Carbohydrate column according to the manufacturer's directions
(Bio-Rad Laboratories, Hercules, Calif., USA). Sucrose, glucose and
fructose are identified refractometrically (Shimadzu RID) by their
retention time and quantified by comparison with sugar
standards.
[0231] The remaining pellet after the hot (68.degree. C.) ethanol:
H.sub.2O extraction is assayed for starch following an overnight
amyloglucosidase treatment and assay of released glucose using the
dinitrosalicylic reagent, as described in Schaffer et al., 1987,
Phytochemistry 26:1883-1887.
[0232] Determination of cellulose and holocellulose content: Dried
plant stem material is ground using a Wiley mill to pass through a
30-mesh screen, and then Soxhlet-extracted with acetone for 24 h.
The extractive free material is used for all further analyses.
Holocellulose and .alpha.-cellulose is determined using a modified
microanalytical method developed by Yokoyama et al. (2002), J Agric
Fd Chem 50: 1040-1044. In short, 200 mg of ground sample is weighed
into a 25-mL round-bottomed flask and placed in a 90.degree. C. oil
bath. The reaction is initiated by the addition of 1 mL of sodium
chlorite solution (400 mg 80% sodium chlorite, 4 mL distilled
water, 0.4 mL acetic acid). An additional 1 mL of sodium chlorite
solution is added every half hour and the sample removed to a cold
water bath after 2 h. The sample is filtered through a coarse
crucible, dried overnight and the holocellulose composition
determined gravimetrically. Fifty milligrams of this dried
holocellulose sample is weighed into a reaction flask and allowed
to equilibrate for 30 min. Four millilitres of 17.5% sodium
hydroxide are added and allowed to react for 30 min, after which 4
mL of distilled water is added. The sample is macerated for 1 min,
allowed to react for an additional 29 min and then filtered through
a coarse filter. Following a 5-min soak in 1.0 M acetic acid, the
sample is washed with 90 mL of distilled water and dried overnight.
The .alpha.-cellulose content is then determined
gravimetrically.
[0233] Results
[0234] Results show that enzyme activity was more than doubled in
some of the transformants, as compared to the non-transformed
control. The results comparing the semi-quantitative expression in
FIG. 8 and the enzyme activity in Table 6 show that there is a good
correlation between the gene expression levels and enzyme activity.
Both gene expression as well as enzyme activity were highest in
lines 4, 10, 11, 23.
TABLE-US-00006 TABLE 6 Enzyme activity in transformed tobacco
plants harboring the UGGPPase gene. Each plant is from an
independent transgenic event. Basal enzyme activity of the
non-transformed tobacco plants is listed as NN. Enzyme activity
(UDPgalactose formed Tobacco line per min per gfw) NN1 6.8 NN2 6.5
UGGP4 10.6 UGGP6 4.0 UGGP10 14.8 UGGP11 14.5 UGGP19 2.4 UGGP21 7.1
UGGP23 13.0 UGGP29 12.3 UGGP37 7.1
[0235] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable
subcombination.
[0236] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims. All
publications, patents and patent applications mentioned in this
specification are herein incorporated in their entirety by
reference into the specification, to the same extent as if each
individual publication, patent or patent application was
specifically and individually indicated to be incorporated herein
by reference. In addition, citation or identification of any
reference in this application shall not be construed as an
admission that such reference is available as prior art to the
present invention.
Sequence CWU 1
1
50117DNAArtificial sequenceSingle strand DNA oligonucleotide
1gcnggnytna artgggt 17217DNAArtificial sequenceSingle strand DNA
oligonucleotide 2ggccanacyt cnacytc 1736PRTArtificial sequenceMelon
UGGPase conserved peptide used for preparation of a degenerate
primer 3Ala Gly Leu Lys Trp Val1 546PRTArtificial sequenceMelon
UGGPase conserved peptide used for preparation of a degenerate
primer 4Glu Val Glu Val Trp Pro1 5517DNAArtificial sequenceSingle
strand DNA oligonucleotide 5tcnagytayc cnggngg 1766PRTArtificial
sequenceMelon UGGPase conserved peptide used for preparation of a
degenerate primer 6Ser Ser Tyr Pro Gly Gly1 5721DNAArtificial
sequenceSingle strand DNA oligonucleotide 7cccaccagca acaagaacaa a
21821DNAArtificial sequenceSingle strand DNA oligonucleotide
8cttcaacccg attggaatgt a 21917DNAArtificial sequenceSingle strand
DNA oligonucleotide 9acnatgggnt gycangg 171017DNAArtificial
sequenceSingle strand DNA oligonucleotide 10gayggntggt ayccncc
171117DNAArtificial sequenceSingle strand DNA oligonucleotide
11ccnccyttna crtcngc 171217DNAArtificial sequenceSingle strand DNA
oligonucleotide 12ccrtcnacyt cyttngg 17136PRTArtificial
sequenceUGPase consensus amino acid sequences 13Thr Met Gly Cys Thr
Gly1 5146PRTArtificial sequenceUGPase consensus amino acid
sequences 14Asp Gly Trp Tyr Pro Pro1 5156PRTArtificial
sequenceUGPase consensus amino acid sequences 15Ala Asp Val Lys Gly
Gly1 5166PRTArtificial sequenceUGPase consensus amino acid
sequences 16Pro Lys Glu Val Asp Gly1 51722DNAArtificial
sequenceSingle strand DNA oligonucleotide 17acattcaacc agagccaata
tc 221822DNAArtificial sequenceSingle strand DNA oligonucleotide
18cacccacaaa ttgttagtgt tg 221921DNAArtificial sequenceSingle
strand DNA oligonucleotide 19gagcansagt gygcnccnga g
212023DNAArtificial sequenceSingle strand DNA oligonucleotide
20gcnccntggt tyttgaanac ctg 232123DNAArtificial sequenceSingle
strand DNA oligonucleotide 21acatcctcgg gggtcaaatc aga
232222DNAArtificial sequenceSingle strand DNA oligonucleotide
22caggcttcgg attcagactt ag 222321DNAArtificial sequenceSingle
strand DNA oligonucleotide 23aacccgattg gaatgtatga t
212421DNAArtificial sequenceSingle strand DNA oligonucleotide
24ccgaagtagc actgtgataa g 212523DNAArtificial sequenceSingle strand
DNA oligonucleotide 25tcctgctctc agtagggata agg 232623DNAArtificial
sequenceSingle strand DNA oligonucleotide 26acatcctcgg gggtcaaatc
aga 232719DNAArtificial sequenceSingle strand DNA oligonucleotide
27gattccgtgc ccagaagtt 192820DNAArtificial sequenceSingle strand
DNA oligonucleotide 28ttccttgctc atcctgtctg 202920DNAArtificial
sequenceSingle strand DNA oligonucleotide 29cacgcaggtt ctccggccgc
203020DNAArtificial sequenceSingle strand DNA oligonucleotide
30tgcgctgcga atcgggagcg 203121DNAArtificial sequenceSingle strand
DNA oligonucleotide 31cagcaataga ctggcaggtg a 213221DNAArtificial
sequenceSingle strand DNA oligonucleotide 32ccaatcgggt tgaagacttg a
2133614PRTCucumis melo 33Met Ala Ser Ser Leu Asp Ser Ala Ala Leu
Thr Leu Ser Asn Leu Ser1 5 10 15Ile Asn Gly Asp Phe Ala Ser Ser Leu
Pro Asn Leu Gln Lys Asn Leu 20 25 30His Leu Leu Ser Pro Gln Gln Val
Glu Leu Ala Lys Ile Leu Leu Glu 35 40 45Leu Gly Gln Ser His Leu Phe
Glu His Trp Ala Glu Pro Gly Val Asp 50 55 60Asp Asn Glu Lys Lys Ala
Phe Phe Asp Gln Val Ala Arg Leu Asn Ser65 70 75 80Ser Tyr Pro Gly
Gly Leu Ala Ser Tyr Ile Lys Thr Ala Arg Gly Leu 85 90 95Leu Ala Asp
Ser Lys Glu Gly Lys Asn Pro Phe Asp Gly Phe Thr Pro 100 105 110Ser
Val Pro Thr Gly Glu Val Leu Thr Phe Gly Asp Asp Ser Phe Val 115 120
125Ser Phe Glu Asp Arg Gly Val Arg Glu Ala Arg Lys Ala Ala Phe Val
130 135 140Leu Val Ala Gly Gly Leu Gly Glu Arg Leu Gly Tyr Asn Gly
Ile Lys145 150 155 160Val Ala Leu Pro Ala Glu Thr Thr Thr Gly Thr
Cys Phe Leu Gln Ser 165 170 175Tyr Ile Glu Tyr Val Leu Ala Leu Arg
Glu Ala Ser Asn Arg Leu Ala 180 185 190Gly Glu Ser Glu Thr Glu Ile
Pro Phe Val Ile Met Thr Ser Asp Asp 195 200 205Thr His Thr Arg Thr
Val Glu Leu Leu Glu Ser Asn Ser Tyr Phe Gly 210 215 220Met Lys Pro
Ser Gln Val Lys Leu Leu Lys Gln Glu Lys Val Ala Cys225 230 235
240Leu Asp Asp Asn Glu Ala Arg Leu Ala Val Asp Pro His Asn Lys Tyr
245 250 255Arg Ile Gln Thr Lys Pro His Gly His Gly Asp Val His Ala
Leu Leu 260 265 270Tyr Ser Ser Gly Leu Leu Lys Asn Trp His Asn Ala
Gly Leu Arg Trp 275 280 285Val Leu Phe Phe Gln Asp Thr Asn Gly Leu
Leu Phe Lys Ala Ile Pro 290 295 300Ala Ser Leu Gly Val Ser Ala Thr
Arg Glu Tyr His Val Asn Ser Leu305 310 315 320Ala Val Pro Arg Lys
Ala Lys Glu Ala Ile Gly Gly Ile Thr Arg Leu 325 330 335Thr His Thr
Asp Gly Arg Ser Met Val Ile Asn Val Glu Tyr Asn Gln 340 345 350Leu
Asp Pro Leu Leu Arg Ala Thr Gly Phe Pro Asp Gly Asp Val Asn 355 360
365Asn Glu Thr Gly Tyr Ser Pro Phe Pro Gly Asn Ile Asn Gln Leu Ile
370 375 380Leu Glu Leu Gly Ser Tyr Ile Glu Glu Leu Ser Lys Thr Gln
Gly Ala385 390 395 400Ile Lys Glu Phe Val Asn Pro Lys Tyr Lys Asp
Ala Thr Lys Thr Ser 405 410 415Phe Lys Ser Ser Thr Arg Leu Glu Cys
Met Met Gln Asp Tyr Pro Lys 420 425 430Thr Leu Pro Pro Ser Ala Arg
Val Gly Phe Thr Val Met Asp Thr Trp 435 440 445Val Ala Tyr Ala Pro
Val Lys Asn Asn Pro Glu Asp Ala Ala Lys Val 450 455 460Pro Lys Gly
Asn Pro Tyr His Ser Ala Thr Ser Gly Glu Met Ala Ile465 470 475
480Tyr Arg Ala Asn Ser Leu Val Leu Arg Lys Ala Gly Val Lys Val Ala
485 490 495Asp Pro Val Glu Gln Val Phe Asn Gly Gln Glu Val Glu Val
Trp Pro 500 505 510Arg Ile Thr Trp Lys Pro Lys Trp Gly Leu Thr Phe
Ser Glu Ile Lys 515 520 525Ser Lys Ile Asn Gly Asn Cys Ser Ile Ser
Pro Arg Ser Thr Leu Val 530 535 540Ile Lys Gly Lys Asn Val Tyr Leu
Lys Asp Leu Ser Leu Asp Gly Thr545 550 555 560Leu Ile Val Asn Ala
Asp Glu Asp Ala Glu Val Lys Val Glu Gly Ser 565 570 575Val His Asn
Lys Gly Trp Thr Leu Glu Pro Val Asp Tyr Lys Asp Thr 580 585 590Ser
Val Pro Glu Glu Ile Arg Ile Arg Gly Phe Arg Ile Asn Lys Ile 595 600
605Glu Gln Glu Glu Arg Asn 610342169DNACucumis melo 34ggcacgaggc
tcaatcgcac ccaacatggc ttcctctctc gattccgctg cactcactct 60ttctaacctt
tccatcaatg gagatttcgc ttcttctctt cccaatttac agaagaatct
120ccaccttcta tctcctcaac aggttgaatt ggcgaagatt ttgttggaat
tggggcagag 180tcatcttttt gagcattggg ccgagcctgg cgttgatgat
aatgaaaaga aggctttctt 240cgaccaggtt gctcggctta attctagcta
tcctgggggg ttggcctcct atatcaagac 300tgccagggga ctcttagcag
attccaaaga aggaaagaac ccatttgatg gcttcactcc 360ctctgttcca
actggtgaag ttttgacttt tggcgatgat agctttgtca gctttgagga
420ccgaggtgta agggaagctc gaaaggctgc atttgttctt gttgctggtg
ggcttgggga 480gcggctagga tataatggaa ttaaggtggc tcttccagca
gaaactacta caggcacatg 540tttcttacag agttacattg aatacgtttt
ggctcttcga gaagccagca atagactggc 600aggtgaaagt gaaacagaga
ttccttttgt tataatgaca tcagatgata ctcatacacg 660tacagtagag
ctgttggaat cgaattccta ttttggaatg aaaccctcac aagttaaact
720tctaaaacag gaaaaagttg cttgtttgga tgataatgag gccaggcttg
ccgttgatcc 780acataacaaa tataggattc agaccaagcc tcatggccat
ggggatgtcc atgcacttct 840gtactctagt ggccttctca aaaattggca
caatgctggt ttaagatggg ttctcttttt 900ccaagataca aatgggcttc
tattcaaggc aattccagct tctttgggtg ttagtgctac 960aagagagtac
catgttaatt ctctagctgt tccacgcaaa gcaaaagaag ccattggtgg
1020aattactcgt cttactcata ctgatgggag gtctatggtt atcaatgtgg
aatataatca 1080gcttgatcca ctgcttagag caactggatt tcccgatggt
gacgtcaata atgagaccgg 1140ctactctcct tttccaggaa atataaatca
actaatttta gaacttggtt cctatattga 1200ggagctgagc aaaacacaag
gtgctataaa ggaatttgtc aatcccaaat ataaagatgc 1260taccaagact
tctttcaagt cttcaacccg attggaatgt atgatgcaag attatccaaa
1320gacattacct ccatcggctc gggttggatt tacggtgatg gatacctggg
ttgcttatgc 1380tccagtgaag aacaaccctg aagatgctgc taaggtaccg
aagggaaacc cttatcacag 1440tgctacttcg ggggaaatgg ccatctaccg
tgcaaatagt cttgttctca gaaaggcagg 1500agttaaagta gccgatccag
ttgaacaggt gttcaatggc caagaggttg aagtctggcc 1560tcgcatcacg
tggaaaccga aatggggttt gaccttttca gagataaaaa gcaaaatcaa
1620tggaaattgc tccatttctc cgcgttctac cttggttatc aaggggaaaa
acgtttatct 1680taaagatctc tccttggatg gaactcttat tgtgaatgca
gatgaagatg ctgaggtaaa 1740agtagagggt tcagtacata acaagggctg
gacactcgaa cccgttgatt ataaagatac 1800ttcagtacca gaagaaataa
ggattagagg gttcagaatc aacaaaatcg agcaggaaga 1860aagaaactga
gcctacaact ttagcctgaa ataccttgaa ggtgaagttg ttatattcat
1920ggcctttatt ggaccagttt ttgctgtgaa ataattcttt ttccttactt
taggaaaagg 1980aaatttgtaa cgattttggt tctataataa atatgtattt
taacgtggcc tggaataatt 2040tgattgagta ataaaaatat tttggagatg
gagaatgaaa tttctttgat acttctcctc 2100acctttattt gaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2160aaaaaaaaa
216935476PRTCucumis melo 35Met Ala Ser Ala Ala Thr Leu Ser Pro Ala
Asp Thr Glu Lys Leu Ser1 5 10 15Lys Leu Lys Ala Ser Val Ser Gly Leu
Thr Gln Ile Ser Glu Asn Glu 20 25 30Lys Ser Gly Phe Ile Asn Leu Val
Ser Arg Tyr Leu Ser Gly Glu Ala 35 40 45Gln His Val Glu Trp Ser Lys
Ile Gln Thr Pro Thr Asp Glu Val Val 50 55 60Val Pro Tyr Asp Ser Leu
Ala Pro Val Pro Asn Asp Pro Ala Glu Thr65 70 75 80Lys Lys Leu Leu
Asp Lys Leu Val Val Leu Lys Leu Asn Gly Gly Leu 85 90 95Gly Thr Thr
Met Gly Cys Thr Gly Pro Lys Ser Val Ile Glu Val Arg 100 105 110Asn
Gly Leu Thr Phe Leu Asp Leu Ile Val Ile Gln Ile Glu Asn Leu 115 120
125Asn Ser Lys Tyr Gly Cys Asn Val Pro Leu Leu Leu Met Asn Ser Phe
130 135 140Asn Thr His Asp Asp Thr Gln Lys Ile Ile Glu Lys Tyr Lys
Gly Ser145 150 155 160Asn Val Asp Ile His Thr Phe Asn Gln Ser Gln
Tyr Pro Arg Leu Val 165 170 175Ala Glu Asp Tyr Leu Pro Leu Pro Ser
Lys Gly Arg Thr Asp Lys Asp 180 185 190Gly Trp Tyr Pro Pro Gly His
Gly Asp Val Phe Pro Ser Leu Lys Asn 195 200 205Ser Gly Lys Leu Asp
Ala Leu Ile Ala Gln Gly Lys Glu Tyr Val Phe 210 215 220Val Ala Asn
Ser Asp Asn Leu Gly Ala Val Val Asp Leu Gln Ile Leu225 230 235
240Asn His Leu Ile Gln Asn Lys Asn Glu Tyr Cys Met Glu Val Thr Pro
245 250 255Lys Thr Leu Ala Asp Val Lys Gly Gly Thr Leu Ile Ser Tyr
Glu Gly 260 265 270Lys Val Gln Leu Leu Glu Ile Ala Gln Val Pro Asp
Glu His Val Asn 275 280 285Glu Phe Lys Ser Ile Gln Lys Phe Lys Ile
Phe Asn Thr Asn Asn Leu 290 295 300Trp Val Asn Leu Lys Ala Ile Lys
Arg Leu Val Glu Ala Asn Ala Leu305 310 315 320Lys Met Glu Ile Ile
Pro Asn Pro Lys Glu Val Asp Gly Ile Lys Val 325 330 335Leu Gln Leu
Glu Thr Ala Ala Gly Ala Ala Ile Arg Phe Phe Asp His 340 345 350Ala
Ile Gly Ile Asn Val Pro Arg Ser Arg Phe Leu Pro Val Lys Ala 355 360
365Thr Ser Asp Leu Leu Leu Val Gln Ser Asp Leu Tyr Thr Leu Val Asp
370 375 380Gly Phe Val Leu Arg Asn Lys Ala Arg Lys Asp Pro Ser Asn
Pro Ser385 390 395 400Ile Glu Leu Gly Pro Glu Phe Lys Lys Val Gly
Asn Phe Leu Ser Arg 405 410 415Phe Lys Ser Ile Pro Ser Ile Ile Glu
Leu Asp Ser Leu Lys Val Val 420 425 430Gly Asp Val Ser Phe Gly Ala
Gly Val Val Leu Lys Gly Lys Val Thr 435 440 445Ile Ser Ala Lys Pro
Gly Thr Lys Leu Ala Val Pro Asp Asn Ala Val 450 455 460Ile Ala Asn
Lys Glu Ile Asn Gly Pro Glu Asp Phe465 470 475361640DNACucumis melo
36atggcatctg ctgctactct tagccctgct gatactgaga agctttccaa acttaaagct
60tctgtttctg gacttaccca gattagtgag aatgagaaat ctggatttat caaccttgtc
120tctcgctatc tcagtgggga agcacagcat gttgaatgga gcaagatcca
gactccaaca 180gatgaagtag tggttcctta tgattccttg gcacctgtac
ctaatgatcc tgctgaaact 240aagaaactat tggacaaact tgttgttttg
aagcttaatg gaggtttggg gaccacaatg 300ggctgcacag gtcctaagtc
agtcattgaa gtccggaatg gtttgacatt tcttgacttg 360attgttatcc
aaatagagaa tcttaattcc aaatatgggt gcaacgttcc tttacttctg
420atgaactcat ttaacactca tgatgatacc caaaagatca ttgaaaaata
caaaggttca 480aatgtggata ttcatacatt caaccagagc caatatccac
gtttggttgc tgaggactat 540cttccactcc ctagcaaagg acgcactgac
aaggatggat ggtaccctcc tggacatggt 600gatgttttcc catccttgaa
aaacagcggc aaacttgatg ccctgatagc tcagggcaag 660gaatatgtct
ttgttgcaaa ctctgacaac ttaggtgccg ttgtggactt gcaaatttta
720aatcatttga tacagaacaa gaatgagtac tgcatggagg tgactcccaa
aaccttggct 780gatgtgaagg gtggtactct tatttcttat gaagggaagg
ttcagttgct tgaaattgct 840caagtccctg atgaacacgt caatgaattc
aagtcaattc agaaattcaa aattttcaac 900actaacaatt tgtgggtgaa
cttgaaagca atcaaaaggc ttgtggaagc caatgcactt 960aagatggaga
ttattccaaa tcccaaggaa gttgatggga ttaaagttct tcagctcgaa
1020acagcagctg gtgcagcaat caggttcttt gatcatgcaa ttggtattaa
tgtgccacga 1080tcacgatttc ttcctgtcaa agcaacttca gatttgcttc
ttgtccagtc tgatctctat 1140actctagttg atggctttgt ccttcgcaac
aaggctagaa aagatccttc caatccttct 1200attgaattgg ggcccgaatt
caagaaggtt ggtaacttcc tgagccgatt caagtcaatt 1260ccgagcatca
ttgaacttga tagccttaaa gtggttggcg atgtttcgtt cggggctggt
1320gtcgttctca aggggaaagt gactatttcg gctaaaccag ggacgaaatt
ggctgtaccc 1380gataacgccg taatagcaaa caaggaaatc aatggcccag
aagatttcta aacaattggt 1440tgcctttcac aactctttca gagcagaaac
cttatggcat ctgcgtaccc tttctctttt 1500aataccaaca aaatcgtgca
gttttgtctg taatatgcgt ccagtttggg aactggtgtt 1560tttgataaga
tgaactttgt tggcattaaa aacaaaatga gtttatatta atatataata
1620cagaataagc aaaagttaag 164037344PRTCucumis melo 37Met Ala Ser
Pro Val Glu Ser Arg Arg Pro Glu Leu Arg Lys Asp Ser1 5 10 15Val Thr
Asn Arg Trp Val Ile Phe Ser Pro Ala Arg Ala Lys Arg Pro 20 25 30Ser
Asp Phe Lys Ser Lys Ser Pro Ala Pro Ser Ser Thr Asp Ser Pro 35 40
45Gln Thr Cys Pro Phe Cys Ile Gly Gln Glu His His Cys Ala Pro Glu
50 55 60Ile Phe Arg Phe Pro Pro Gln Asn Pro Asp Trp Lys Val Arg Val
Ile65 70 75 80Gln Asn Leu Tyr Pro Ala Leu Ser Arg Asp Lys Asp Leu
Asp Ser Ser 85 90 95Thr Ser Leu Ser Ser Gly Ser Leu Leu Trp Gly Cys
Leu Leu Asp Gly 100 105 110Tyr Gly Phe His Asp Val Ile Ile Glu Ser
Pro Val His Ser Val His
115 120 125Leu Ser Asp Leu Thr Pro Glu Asp Val Ala Gln Val Leu Phe
Ala Tyr 130 135 140Lys Lys Arg Ile Leu Gln Leu Ala Ser Asp Asp Ser
Ile Lys Tyr Val145 150 155 160Gln Val Phe Lys Asn His Gly Ala Ser
Ala Gly Ala Ser Met Thr His 165 170 175Pro His Ser Gln Met Val Gly
Leu Pro Val Ile Pro Pro Ser Val Thr 180 185 190Thr Arg Leu Asp Ser
Met Lys Gln Tyr Phe Asn Glu Thr Gly Lys Cys 195 200 205Ser Ile Cys
His Val Pro Thr Lys Asp Leu Leu Val Asp Glu Ser Val 210 215 220His
Phe Ile Ser Val Val Pro Tyr Ala Ala Ser Phe Pro Phe Glu Leu225 230
235 240Trp Ile Val Pro Arg Asp His Val Ser His Phe His Glu Leu Asp
Gln 245 250 255Glu Lys Ala Val Asp Leu Gly Gly Leu Leu Lys Val Thr
Leu Ile Lys 260 265 270Met Ser Leu Gln Leu Asn Lys Pro Pro Phe Asn
Phe Met Ile His Thr 275 280 285Ser Pro Leu Gln Ala Ser Asp Ser Asp
Leu Ala Tyr Ser His Trp Phe 290 295 300Phe Gln Ile Val Pro His Leu
Ser Gly Val Gly Gly Phe Glu Leu Gly305 310 315 320Thr Gly Cys Tyr
Ile Asn Pro Val Phe Pro Glu Asp Ala Ala Lys Val 325 330 335Met Arg
Glu Val Asn Ile Ser Ile 340381500DNACucumis melo 38ctaacaacgt
tctaggctat ccggtcagaa tttctttcag cctttttcgc cgtggaaaat 60ggcgtcgccg
gttgaatctc gccgtcccga actccggaag gactcagtaa ctaatcgttg
120ggtcatattc tcacctgctc gagctaaacg accctccgat ttcaaatcca
aatccccagc 180cccttcttca actgattctc ctcaaacatg ccccttctgc
attggccaag agcaccactg 240cgctcccgag atctttcgat ttcctcctca
gaaccccgac tggaaagttc gcgttattca 300aaatctctat cctgctctca
gtagggataa ggatctcgat tcttcaactt ccctgagctc 360cggttcactc
ttatggggtt gccttttgga cgggtatggg ttccacgacg tcatcattga
420gtctcctgtt cactcagttc atctctctga tttgaccccc gaggatgtcg
ctcaagttct 480ttttgcgtat aagaagcgga ttctgcagct cgcaagcgat
gacagcatca aatatgttca 540ggtgtttaaa aaccatggtg cctcagctgg
ggcatcaatg acgcaccccc acagtcagat 600ggtgggtctt ccagtcattc
ctccctctgt tactactcga cttgatagta tgaagcagta 660tttcaatgag
acggggaaat gtagcatttg tcatgttcct acaaaggacc ttttggttga
720tgaatcagtc catttcattt ctgttgttcc ctatgcagcc tcgtttccgt
ttgagctctg 780gatcgttccc cgtgaccatg tttctcattt tcatgagcta
gaccaggaga aggctgttga 840tcttggaggg ctattgaaag tgacactcat
aaagatgtct ctgcagctga acaaaccacc 900attcaacttc atgattcaca
cttctccctt gcaggcttcg gattcagact tagcttacag 960ccactggttt
tttcagattg ttcctcacct ttctggtgta ggggggtttg aactaggaac
1020tggttgctac atcaatcctg tttttccaga ggatgctgct aaagtcatga
gggaggttaa 1080catttctata taggctcagg cccaggtacg ttttattctg
atgaaaaatc tctccctttt 1140ctttctggtg tgagattaga actggcctac
ctttttcctt atgaataggt taacactagt 1200tttttttttt tcctgtaaaa
aatctgtcat aacttcactt gggatgtttc tgggaaatcg 1260ttcgtgagta
atggctactt gtttgttgat gattctccta tttggccctg aaagcgagat
1320ctcttacttt gaaatctatt gtgcacagga tatatattac ctaggtagca
caagtacaat 1380aatgagatgg acgcaacatg acatggacat ggtaacacac
tctttaacat atatcatttt 1440tatacatttt gatgaaaact ggatattaat
tttgattttg tttgaaaact ttgcaattaa 15003914PRTArtificial sequenceA
peptide sequence obtained from the peptide microsequencing of the
purified melon UGGPase 39Leu Asn Ser Ser Tyr Pro Gly Gly Leu Ala
Ser Tyr Ile Lys1 5 104014PRTArtificial sequenceA peptide sequence
obtained from the peptide microsequencing of the purified melon
UGGPase 40Leu Thr Phe Gly Asp Asp Ser Phe Val Ser Phe Glu Asp Arg1
5 104126PRTArtificial sequenceA peptide sequence obtained from the
peptide microsequencing of the purified melon UGGPase 41Trp Val Leu
Phe Phe Gln Asp Thr Asn Gly Leu Leu Phe Lys Ala Ile1 5 10 15Pro Ala
Ser Leu Gly Val Ser Ala Thr Arg 20 254223PRTArtificial sequenceA
peptide sequence obtained from the peptide microsequencing of the
purified melon UGGPase 42Ser Pro Phe Pro Gly Asn Ile Asn Gln Leu
Ile Leu Glu Leu Gly Ser1 5 10 15Tyr Ile Glu Glu Leu Ser Lys
20439PRTArtificial sequenceA peptide sequence obtained from the
peptide microsequencing of the purified melon UGGPase 43Lys Val Ala
Asp Pro Val Glu Gln Val1 5447PRTArtificial sequenceA peptide
sequence obtained from the peptide microsequencing of the purified
melon UGGPase 44Glu Val Glu Val Trp Pro Arg1 5459PRTArtificial
sequenceA peptide sequence obtained from the peptide
microsequencing of the purified melon UGGPase 45Trp Gly Leu Thr Phe
Ser Glu Ile Lys1 546877DNAArtificial sequenceCauliflower mosaic
virus 35S promoter 46aagcttgcat gcctgcaggt ccccagatta gccttttcaa
tttcagaaag aatgctaacc 60cacagatggt tagagaggct tacgcagcag gtctcatcaa
gacgatctac ccgagcaata 120atctccagga aatcaaatac cttcccaaga
aggttaaaga tgcagtcaaa agattcagga 180ctaactgcat caagaacaca
gagaaagata tatttctcaa gatcagaagt actattccag 240tatggacgat
tcaaggcttg cttcacaaac caaggcaagt aatagagatt ggagtctcta
300aaaaggtagt tcccactgaa tcaaaggcca tggagtcaaa gattcaaata
gaggacctaa 360cagaactcgc cgtaaagact ggcgaacagt tcatacagag
tctcttacga ctcaatgaca 420agaagaaaat cttcgtcaac atggtggagc
acgacacact tgtctactcc aaaaatatca 480aagatacagt ctcagaagac
caaagggcaa ttgagacttt tcaacaaagg gtaatatccg 540gaaacctcct
cggattccat tgcccagcta tctgtcactt tattgtgaag atagtggaaa
600aggaaggtgg ctcctacaaa tgccatcatt gcgataaagg aaaggccatc
gttgaagatg 660cctctgccga cagtggtccc aaagatggac ccccacccac
gaggagcatc gtggaaaaag 720aagacgttcc aaccacgtct tcaaagcaag
tggattgatg tgatatctcc actgacgtaa 780gggatgacgc acaatcccac
tatccttcgc aagacccttc ctctatataa ggaagttcat 840ttcatttgga
gagaacacgg gggactctag aggatcc 877472322DNAArtificial
sequenceArabidopsis At6669 promoter 47aagctttaag ctccaagccc
acatctatgc acttcaacat atctttttct agatgagttg 60gtaaaagtag aaaaagatat
gatgatttta aatttgtttc tatttatatg tgttcatcga 120aacttcattt
tttttagttt taatagagag tttatatgac ttttaaaaat tgatttaaaa
180ctgtgtcaaa aattaaaagg acaataaaaa atttgcatac aaccgaaaat
acttatattt 240agacaagaaa aaataatact tgtgatgctg attttatttt
attatatatc atgaatcatg 300atcatccaat tttccggata agccaaagtc
aaaatgatgg gttcccccta atcttttatg 360ctgagaaata gatgtatatt
cttagatagt aatataaaat tgggttaaag aatgatgatt 420cgattatagc
ctcaactaga agatacgtgt agtgcaggtg tgtagttaac tggtggtagt
480ggcagacaac cagattagga gttaaataaa gcctttagat ttgagagatt
gaaatattcg 540attggaacct ttctagattt ttacagccat ctaaaattag
atgcagatca cctactacca 600ttcaaaaatg aacaaaataa tttcatttac
attttcctag cataagatat aataataaaa 660tagtgctcat tttaattact
ttttctaaat attttcgtta ttttaaattt tgcttgtcta 720tactctacag
ctcatttaat aacggaaaca aaaataattg cagggatacg gatgggtagc
780tttcaaaact tacatcatct tctgtttctt gagatcaact atttttggag
ctttgtctca 840atcgtaccaa aggataatgg tcctacctcc ttttgcattc
ttaactttat cttctctact 900tatttctttt ttgggatttt tgggggtatt
attttatctt ttgtagatat acacattgat 960ttactacaaa cgtatactac
tatccatctt caactcttcg gaatatgatt tcgaaaaaac 1020tatgaagatt
aacgggtatc ttaaacatgt taagatacac cggacaattt tcatttagaa
1080gaattgatat gcaattaaca ataaatagtt gatgatcttt tagttttgaa
gatgtgcgtt 1140aagacttaag cgtgtggtaa caaggtggga ctcgggcaac
gcaaagcctt gtagagtcca 1200cttgctcaac ttgtctttct tttatctctt
ttccaagtct caagattcaa tgaactccgt 1260gtaacacaaa cacgcccata
gatgagctca tttttggtat ttccaatatt gccactccat 1320gataatatca
tctagggatg gggttcattt attttgaaat ctcaacaaat ctcgtcgatt
1380ctaacacaca tgattgattt gtttacttac ttgaaagttg gcaactatct
gggattaaaa 1440tttatctttt tctactgcta gctagaagca tctatatatg
ttagcctaat acgtggaaga 1500tgtcattgct aataatggct aaagatgtgt
attaattttt cttctttttt ccttgaattt 1560ttgttctttg acataaacta
tgctgtcaaa atgtgtagaa tctttttaca taaatcattc 1620cctgttacac
actaaaaggt tcacaacgga cgattgtatt ggacttccag atcataaacc
1680atgcaaaact gaaaaccaca agaataatta gttctaactt tagaacgttc
gtacgtgttt 1740catgttcaaa aagcgtcaat tataaaagtt gggaaattac
ttttgagttt tgacatttct 1800aaggacagtc aaatatgaca acattgggat
gcaacttacc ttgtattaac ttattttgtt 1860ataaaaccat atattacata
ttttaaaggg ttgataaata atcaaatata ccaaaacata 1920gcttttcaat
atatttgtaa aacacgtttg gtctactagc taattatgag aacatttgtt
1980caatgcatga ttatctagta tctactagtg gattatgaaa attagatatt
ttcattgcat 2040gattatcttc catatatagt gataacatca aaagaatcta
caccaattat tgcatttttt 2100cattatataa taagcactaa actgtaaaat
tatattcagc cacccaaacc atgacaaatc 2160accttaaagg cttaaacaca
taacagccat tacgagtcac aggtaagggt ataatagtaa 2220agaatcaatc
tatataatat acgacccacc ctttctcatt ctttctggag agtaacatcg
2280agacaaagaa gaaaaactaa aaaagagaac cccaaaggat cc
232248976DNAArtificial sequenceMaize ubiquitin gene derived
promoter 48gtgcagcgtg acccggtcgt gcccctctct agagataatg agcattgcat
gtctaagtta 60taaaaaatta ccacatattt tttttgtcac acttgtttga agtgcagttt
atctatcttt 120atacatatat ttaaacttta ctctacgaat aatataatct
atagtactac aataatatca 180gtgttttaga gaatcatata aatgaacagt
tagacatggt ctaaaggaca attgagtatt 240ttgacaacag gactctacag
ttttatcttt ttagtgtgca tgtgttctcc tttttttttg 300caaatagctt
cacctatata atacttcatc cattttatta gtacatccat ttagggttta
360gggttaatgg tttttataga ctaatttttt tagtacatct attttattct
attttagcct 420ctaaattaag aaaactaaaa ctctatttta gtttttttat
ttaataattt agatataaaa 480tagaataaaa taaagtgact aaaaattaaa
caaataccct ttaagaaatt aaaaaaacta 540aggaaacatt tttcttgttt
cgagtagata atgccagcct gttaaacgcc gtcgacgagt 600ctaacggaca
ccaaccagcg aaccagcagc gtcgcgtcgg gccaagcgaa gcagacggca
660cggcatctct gtcgctgcct ctggacccct ctcgagagtt ccgctccacc
gttggacttg 720ctccgctgtc ggcatccaga aattgcgtgg cggagcggca
gacgtgagcc ggcacggcag 780gcggcctcct cctcctctca cggcacggca
gctacggggg attcctttcc caccgctcct 840tcgctttccc ttcctcgccc
gccgtaataa atagacaccc cctccacacc ctctttcccc 900aacctcgtgt
tgttcggagc gcacacacac acaaccagat ctcccccaaa tccacccgtc
960ggcacctccg cttcaa 97649913DNAArtificial sequenceRice actin gene
derived promoter 49tcgaggtcat tcatatgctt gagaagagag tcgggatagt
ccaaaataaa acaaaggtaa 60gattacctgg tcaaaagtga aaacatcagt taaaaggtgg
tataaagtaa aatatcggta 120ataaaaggtg gcccaaagtg aaatttactc
ttttctacta ttataaaaat tgaggatgtt 180ttgtcggtac tttgatacgt
catttttgta tgaattggtt tttaagttta ttcgcgattt 240ggaaatgcat
atctgtattt gagtcggttt ttaagttcgt tgcttttgta aatacagagg
300gatttgtata agaaatatct ttaaaaaacc catatgctaa tttgacataa
tttttgagaa 360aaatatatat tcaggcgaat tccacaatga acaataataa
gattaaaata gcttgccccc 420gttgcagcga tgggtatttt ttctagtaaa
ataaaagata aacttagact caaaacattt 480acaaaaacaa cccctaaagt
cctaaagccc aaagtgctat gcacgatcca tagcaagccc 540agcccaaccc
aacccaaccc aacccacccc agtgcagcca actggcaaat agtctccacc
600cccggcacta tcaccgtgag ttgtccgcac caccgcacgt ctcgcagcca
aaaaaaaaaa 660aagaaagaaa aaaaagaaaa agaaaaacag caggtgggtc
cgggtcgtgg gggccggaaa 720agcgaggagg atcgcgagca gcgacgaggc
ccggccctcc ctccgcttcc aaagaaacgc 780cccccatcgc cactatatac
ataccccccc ctctcctccc atccccccaa ccctaccacc 840accaccacca
ccacctcctc ccccctcgct gccggacgac gagctcctcc cccctccccc
900tccgccgccg ccg 913501172DNAArtificial sequenceSynthetic Super
MAS promoter 50tacaggccaa attcgctctt agccgtacaa tattactcac
cggtgcgatg ccccccatcg 60taggtgaagg tggaaattaa tgatccatct tgagaccaca
ggcccacaac agctaccagt 120ttcctcaagg gtccaccaaa aacgtaagcg
cttacgtaca tggtcgataa gaaaaggcaa 180tttgtagatg ttaacatcca
acgtcgcttt cagggatccc gaattccaag cttggaattc 240gggatcctac
aggccaaatt cgctcttagc cgtacaatat tactcaccgg tgcgatgccc
300cccatcgtag gtgaaggtgg aaattaatga tccatcttga gaccacaggc
ccacaacagc 360taccagtttc ctcaagggtc caccaaaaac gtaagcgctt
acgtacatgg tcgataagaa 420aaggcaattt gtagatgtta acatccaacg
tcgctttcag ggatcccgaa ttccaagctt 480ggaattcggg atcctacagg
ccaaattcgc tcttagccgt acaatattac tcaccggtgc 540gatcccccca
tcgtaggtga aggtggaaat taatgatcca tcttgagacc acaggcccac
600aacagctacc agtttcctca agggtccacc aaaaacgtaa gcgcttacgt
acatggtcga 660taagaaaagg caatttgtag atgttaacat ccaacgtcgc
tttcagggat cccgaattcc 720aagcttgggc tgcaggtcaa tcccattgct
tttgaagcag ctcaacattg atctctttct 780cgagggagat ttttcaaatc
agtgcgcaag acgtgacgta agtatccgag tcagttttta 840tttttctact
aatttggtcg tttatttcgg cgtgtaggac atggcaaccg ggcctgaatt
900tcgcgggtat tctgtttcta ttccaacttt ttcttgatcc gcagccatta
acgacttttg 960aatagatacg ctgacacgcc aagcctcgct agtcaaaagt
gtaccaaaca acgctttaca 1020gcaagaacgg aatgcgcgtg acgctcgcgg
tgacgccatt tcgccttttc agaaatggat 1080aaatagcctt gcttcctatt
atatcttccc aaattaccaa tacattacac tagcatctga 1140atttcataac
caatctcgat acaccaaatc ga 1172
* * * * *
References