U.S. patent application number 11/626131 was filed with the patent office on 2007-06-07 for method of producing transgenic plants having improved amino acid composition and improved yielding.
This patent application is currently assigned to AJINOMOTO CO. INC.. Invention is credited to Takao Kida, Hiroaki KISAKA.
Application Number | 20070130643 11/626131 |
Document ID | / |
Family ID | 24932772 |
Filed Date | 2007-06-07 |
United States Patent
Application |
20070130643 |
Kind Code |
A1 |
KISAKA; Hiroaki ; et
al. |
June 7, 2007 |
METHOD OF PRODUCING TRANSGENIC PLANTS HAVING IMPROVED AMINO ACID
COMPOSITION AND IMPROVED YIELDING
Abstract
The object of the present invention is to provide transgenic
plants which accumulate free amino acids, particularly at least one
amino acid selected from among glutamic acid, asparagine, aspartic
acid, serine, threonine, alanine and histidine accumulated in a
large amount, in the edible parts thereof, and a method of
producing them are provided. The other object of the present
invention is to provide a method of increasing the yielding of
potato and to provide a transgenic potato of which yielding can be
increased. In the present invention, a sequence encoding glutamate
dehydrogenase (GDH) gene is introduced into a plant together with a
suitable regulatory sequence to express it in a plant cell, and
thereby the GDH gene is excessively expressed.
Inventors: |
KISAKA; Hiroaki; (Kanagawa,
JP) ; Kida; Takao; (Kanagawa, JP) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND, MAIER & NEUSTADT, P.C.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Assignee: |
AJINOMOTO CO. INC.
Tokyo
JP
|
Family ID: |
24932772 |
Appl. No.: |
11/626131 |
Filed: |
January 23, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10734282 |
Dec 15, 2003 |
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11626131 |
Jan 23, 2007 |
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PCT/JP01/05077 |
Jun 14, 2001 |
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10734282 |
Dec 15, 2003 |
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09729821 |
Dec 6, 2000 |
6969782 |
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PCT/JP01/05077 |
Jun 14, 2001 |
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Current U.S.
Class: |
800/278 ;
800/317.2; 800/317.4 |
Current CPC
Class: |
C12Y 104/01002 20130101;
C12N 9/93 20130101; Y02A 40/146 20180101; C12N 9/0004 20130101;
C12Y 104/01004 20130101; C12N 9/0016 20130101; C12N 15/8241
20130101; C12N 15/8251 20130101; C12N 15/8261 20130101 |
Class at
Publication: |
800/278 ;
800/317.2; 800/317.4 |
International
Class: |
A01H 5/00 20060101
A01H005/00; A01H 1/00 20060101 A01H001/00 |
Claims
1-19. (canceled)
20. A method of producing a transgenic potato increased in the
total weight of the tuber parts compared to a untransfonned potato
cultured under the same conditions, which comprises the step of
transfonring a plant with a genetic construct causing excessive
expression of a glutamate dehydrogenase (GDH) gene, and selecting
or identifying the transformed plant based on a character imparted
by a marker gene connected to the genetic construct, screening the
transformed plant in which the total weight of the tuber parts is
increased and selecting the transformed plant in which the total
weight of the tuber parts is increased.
21. The method according to claim 20, wherein the total weight of
the tuber parts of the potato exhibits 1.5-fold or more increase
compared to a untransformed potato cultured under the same
conditions.
22. The method according to claim 20, wherein the genetic construct
contains a gene encoding GDH functionally connected to a powerful
constitutive promoter.
23. The method according to claim 20, wherein the constitutive
promoter is CaMV35S promoter.
24. The method according to claim 20, wherein the genetic construct
contains Aspergillus nidulans NADP-GDH gene.
25. The method according to claim 20, wherein GDH contains a
transit peptide for mitochondria.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates to transgenic plants having an
increased free amino acid content, and a method of producing them.
In particular, the present invention relates to transgenic plants
containing at least one of asparagine, aspartic acid, serine,
threonine, alanine, histidine and glutamic acid accumulated in a
large amount, and a method of producing them and also the present
invention relates to a method of increasing an yield of potato, a
transgenic potato of which yielding may be increased and a method
of producing the transgenic potato.
[0002] The technique of transforming a plant by introducing a
specified gene was firstly reported in the world in the study where
it was achieve by introducing a gene into tobacco with
Agrobacterium tumefaciens, a soil microorganism. Thereafter, many
products having useful agricultural features were produced, and it
was also tried to let plants produce useful components. A plant
breeding method by such a transgenic plants producing technique is
considered to be hopeful in place of the ordinary, traditional
breeding technique, such as cross-fertilization. Among them,
improvement in the characteristics of plants concerning nitrogen
assimilation is also being studied. Particularly, the study of
amino acids which are encompassed in the products is particularly
prospering because they are important ingredients in fruits,
tubers, roots of root crops and seeds and also they exert a great
influence on the tastes.
[0003] Reports on the biosynthesis of amino acids include, for
example, a report that free lysine content of tobacco was increased
to 200 times as high content by introducing E. coli
dihydrodipicolinate synthase DHDPS gene into tobacco (U.S. Pat. No.
5,258,300, Molecular Genetics Res. & Development); a report
that free lysine content was increased by introducing aspartate
kinase AK gene (EP 485970, WO 9319190); a report that asparagine
content was increased to 100 times as high content by introducing
asparagine synthetase AS gene into tobacco (WO 9509911, Univ. New
York, WO 9013533, Univ. Rockfeller); and a report that tryptophan
content was increased to 90 times as high content by introducing an
anthranilic acid-synthesizing enzyme into a rice plant (WO 9726366,
DEKALB Genetic Corp). The plants into which a gene is incorporated
are not limited to model plants such as tobacco and Arabidopsis
thaliana but plants which produce fruits such as tomato are also
used. For example, as for tomatoes, a transformant thereof was
obtained by Agrobacterium co-cultivation method in 1986 [S.
McCormick, et. al., Plant Cell Reports, 5, 81-84 (1986); Chyi, et.
al., Mol. Gen. Genet., 204, 64-69 (1986)]. Since then,
investigations were made for the improvement of the transforming
system. Various genes relating to the biosynthesis of amino acids
and nitrogen assimilation other than those described above are also
known. They include asparaginase and glutamine synthase (GOGAT),
and the nucleotide sequences of them were also reported.
[0004] Glutamic acid which is a kind of .alpha.-amino acids is
widely distributed in proteins and is used for seasonings. It is
known that a tasty component of tomato and also a tasty component
of fermentation products of soybeans (such as soy sauce and
fermented soy paste) are all glutamic acid. It is also known that
glutamic acid is synthesized in the first step of nitrogen
metabolism in higher plants. It is also known that glutamine and
asparagine generated from glutamic acid are distributed to tissues
through phloems and used for the synthesis of other amino acids and
proteins. It was reported that in plants, photosynthesis products
such as sucrose and amino acids are present in a high concentration
in the fluid of phloems which are the transporting pathway of the
products [Mitsuo Chino et al., "Shokubutsu Eiyo/Hiryogaku" p. 125
(1993)]. As the example where the photosynthesis products are
contained in a high concentration in edible parts of plants, it is
known that about 0.25 g/100 g f. w. of the photosynthesis products
is contained in tomato fruits ["Tokimeki" No. 2, Nippon Shokuhin
Kogyo Gakkaishi, Vol. 39, pp. 64-67 (1992)]. However, it can not be
easily conducted to accumulate glutamic acid in a high
concentration in plant bodies, because it is a starting material
for amino group-donors and also it is metabolized in various
biosynthetic pathways as described above even though the
biosynthesizing capacity of the source organs may be improved. As
far as the applicant knows, it has never been succeeded to
remarkably increase glutamic acid concentration in plants by either
mated breeding or gene manipulation.
[0005] The first step of the assimilation of inorganic nitrogen
into an organic substance is mainly the incorporation of ammonia
into glutamic acid to generate glutamine, which is catalyzed by
glutamine synthase (GS). Then glutamine, with .alpha.-ketoglutaric
acid, are catalyzed by glutamate synthase (GOGAT) and generate 2
molecules of glutamic acid. This GS/GOGAT cycle is considered to be
the main pathway of nitrogen assimilation in plants [Miflin and
Lea, Phytochemistry 15; 873-885 (1976)]. On the other hand, it is
also known that the ammonia assimilation proceeds also through a
metabolic pathway other than the pathway wherein ammonia is
incorporated by being catalyzed by GS [Knight and Langston-Unkefer,
Science, 241: 951-954 (1988)]. Namely, ammonia is incorporated into
.alpha.-ketoglutaric acid to form glutamic acid, which is catalyzed
by glutamate dehydrogenase (GDH). However, plant GDH has a high Km
value for ammonia. The role of this pathway under normal growing
conditions has not yet been elucidated enough because ammonia is
toxic and the concentration of intracellular ammonia is usually
low. It is reported in an investigation that the ammonia
contributes to the nitrogen assimilation when ammonium
concentration in the cells is increased over a normal level (Knight
and Langston-Unkefer, supra.).
[0006] In plants, glutamate dehydrogenase (GDH) catalyzes the
reversible reaction in which ammonia is released from glutamic acid
to generate .alpha.-ketoglutaric acid, adversely, ammonia is
incorporated into .alpha.-ketoglutaric acid to generate glutamic
acid as discussed above. It is considered that the former occurs
when the content of ammonia nitrogen is high, and the latter occurs
when nitrate nitrogen content is high [Robinson et al., Plant
Physiol. 95; 809-816 (1991); and Robinson et al., Plant Physiol.
98; 1190-1195 (1992)]. The directionality of this enzyme is not
clear, unlike GDH-A enzyme which acts in microorganisms to
synthesize glutamic acid or GDH-B enzyme which acts on them to
decompose it. In plants, it is considered that there are two kinds
of such enzymes, i.e. NADP-depending GDH which functions in
chloroplasts and NAD-dependent GDH which functions in
mitochondrias. Since GDH has a high Km value for ammonia and it is
highly related to the ammonia level during the photorespiration, it
is supposed that NAD-dependent GDH localized in mitochondria has an
important role in the assimilation of ammonia [Srivastava and Singh
RP, Phytochemistry, 26; 597-610 (1987).
[0007] It is known that plant GDH comprises a hexamer composed of
two different kinds of polypeptides (.alpha.-subunits and
.beta.-subunits) linked with each other at random and that there
are seven isozyme patterns depending on the degree of the linkage.
After investigations wherein grapevine calli were used, the
following facts were reported: When calli cultured in a medium
containing a nitrate and glutamic acid were subjected to
electrophoresis, an isozyme comprising .beta.-subunits was
increased on the cathodic side. On the contrary, when calli
cultured in a medium containing ammonia and glutamine were
subjected to the electrophoresis, an isozyme comprising
.alpha.-subunits was increased on the anodic side. Further, when
the calli were transferred from the nitrate medium into the ammonia
medium, GDH activity was increased 3-fold as high activity
(.alpha.-subunits were increased 4-fold and .beta.-subunits were
decreased), the activity was moved from the cathod region to the
anode region [Loulakakis and Poubelakis--Angelakis, Plant Physiol.
97; 104-1111 (1996)]. According to this report, .alpha.-subunits
were considered to play an important role in the assimilation of
ammonia.
[0008] In 1995, Sakakibara et al. [Plant Cell Physiol., 33;
1193-1198] isolated GDH gene of a plant for the first time from two
isozyme bands on the cathodic region in seven isozyme bands in
maize roots. Thereafter, GDH genes were isolated from grapevine
[Syntichaki et al., Gene 168: 87-92 (1996)], Arabidopsis
[Melo-Olivera et al., Proc. Natl. Acad. Sci., USA 93; 4718-4723
(1996)] and tomato [Purnell et al., Gene 186; 249-254 (1997)]. In
particular, the genes isolated from grapevine calli were isolated
from an isozyme expressed in ammonia-treated cells, and they are
considered to be the genes encoding .alpha.-subunits. All the genes
contained a transit peptide which is functional in mitochondria.
GDH genes of maize or tomato are expressed in a large amount in the
root, while those of Arabidopsis are expressed in the leaves and
flowers. It was reported that only one copy of gene is present in
tomato, while two or more genes are present in maize, Arabidopsis
and grapevine, which suggested that the constitution and function
of genes are different among plants and complicated.
[0009] Transgenic plants into which said GDH gene was introduced
were also produced. It was reported that when glutamate
dehydrogenase GDH (NADP-GDH) gene from Escherichia coli was
introduced into tobacco and maize for the purpose of imparting
resistance to phosphinothricin used as a herbicide, the glutamic
acid content of the roots of them was increased to 1.3 to 1.4 times
as high [Lightfoot David et al, CA 2180786 (1966)]. According to
this report, glutamic acid content of tobacco roots was increased
from 14.7 mg/100 gf.w. to 20.6 mg/100 gf. w., and that of maize
roots was increased from 16.2 mg/100 gf. w. to 19.1 mg/100 gf. w.
Additionally, Lightfoot et al. reported that the glutamic acid
content was significantly reduced in seeds of the transformed maize
into which GDH gene had been introduced (U.S. Pat. No. 5,998,700).
Although there are other reports on the use of GDH gene, no example
is given therein [WO 9509911, .alpha., .beta.-subunits from
chlorella (WO 9712983)]. In addition, no analytical value of amino
acids of glutamic acid group was given therein.
[0010] Additionally, photosynthetic phosphoenolpyruvate carboxylase
(C.sub.4-PEPC) gene and NADP-dependent malate dehydrogenase (cpMDH)
gene (Beaujean et al., Plant Science 160:1199-1210 (2001)),
GA20-oxidase gene (Carrera et al., Plant J. 22: 247-256 (2000)),
Hexokinase 1 gene (Veramendi et al., Plant Physioil 121: 123-134
(1999)), NADP-dependent cytosolic isocitrate dehydrogenase (Kruse
et al., Planta 205: 82-91 (1998)), ADP glucose pyrophosphorylase
gene (Greene et al., Proc Natl. Acad. Sci. USA 93 (1996):
1509-1513; Rober et al., Planta 199: 528-536 (1996)), or cytosolic
fructose-1,6-bisphosphatase gene (Zrenner et al., Plant J. 9:
671-681 (1996)) was introduced into potatoes, in order to modify
the starch content in potato tubers, but the yield of potato tubers
was not substantially affected. Redros et al. (Planta 209: 153-160
(1999)) reported that the number of tubers was increased when
S-adenosylmethionine decarboxylase gene was introduced into
potatoes and over expressed, but the size of tubers was small and
the total weight of tubers did not differ from the total weight of
non-transformants. Additionally, Van Asshce et al. (U.S. Pat. No.
5,981,952) reported that sucrose phosphate synthase (SPS) gene was
introduced to a potato and the yield of the potato tubers was
investigated under various concentration of CO.sub.2, which
resulted in the increase of dry weight of a tuber. However, the
amount of increase was very small. As far as the applicant knows,
it has never been succeeded to remarkably increase the yield of
potato tubers.
SUMMARY OF THE INVENTION
[0011] The object of the present invention is to provide a method
of increasing free amino acid contents of storage organs of plants,
in particular, at least one of glutamic acid, asparagine, aspartic
acid, serine, threonine, alanine and histidine contained in edible
parts including roots, tubers, fruits and seeds of plants; and also
to provide transgenic plants in which free amino acids are
accumulated in a large amount.
[0012] Another object of the present invention is to provide a
method of increasing the yield of potato and to provide a
transgenic potato of which yield is increased.
[0013] The object of the present invention is attained by providing
a plant having a changed expression levels and/or expression
balance of organ-specific expression of major enzymes concerning
the assimilation and utilization of nitrogen, and providing a
method of producing such a plant. Such plants can be produced by
introducing at least one gene encoding an enzyme which assimilates
or utilizes nitrogen together with a suitable regulatory sequence
and excessively expressing the same or repressing the
expression.
[0014] The transgenic plant in which a free amino acid is
accumulated in a large amount according to the present invention,
particularly a plant in which at least one of glutamic acid,
asparagine, aspartic acid, serine, threonine, alanine and
histidine, particularly glutamic acid, is accumulated in a large
amount, may be obtained by introducing a glutamate dehydrogenase
(GDH) gene derived from eucaryote together with a suitable
regulatory sequence into the plant and overexpress the gene.
[0015] The potato being increased in the yield according to the
present invention may be also obtained by introducing a glutamate
dehydrogenase (GDH) gene derived from eucaryote together with a
suitable regulatory sequence to the plant and overexpress the
gene.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 shows the comparison of the nucleotide sequence of
glutamate dehydrogenase (GDH) AN-gdh-17 gene from Aspergillus
nidulans with the known nucleotide sequence of NADP-GDH gene. The
upper line shows the nucleotide sequence of NADP-GDH gene, and the
lower line shows that of AN-gdh-17 gene.
[0017] FIG. 2 shows the comparison of the nucleotide sequence of
glutamate dehydrogenase (GDH) AN-gdh-17 gene from Aspergillus
nidulans with the known nucleotide sequence of NADP-GDH gene
(continued from FIG. 1). The upper line shows the nucleotide
sequence of NADP-GDH gene, and the lower line shows that of
AN-gdh-17 gene.
[0018] FIG. 3 shows the cloning strategy of AN-gdh-17 gene into Ti
plasmid (pMAT037). In the figures, 35S Pro represents CaMV 35S
promoter, and Term represents a terminator.
[0019] FIG. 4 is a schematic view showing the strategy of
constructing a genetic construct containing the sequence encoding
AN-gdh-17 gene from Aspergillus connected to the transit peptide,
i. e. pCt-AN-gdh, pCt-dAN-gdh and pMt-dAN-gdh, and the structures
of them.
[0020] FIG. 5 is a schematic diagram of the strategy for removing
the splicing region from AN-gdh-17 gene, wherein thick lines show
the splicing region, and P1 through P4 each represent a PCR
primer.
[0021] FIG. 6 is a schematic diagram showing the strategy for
connecting the transit peptide sequence to Aspergillus AN-gdh-17
gene sequence, wherein A represents the nucleotide sequence of the
transit peptide, B represents the nucleotide sequence of AN-gdh-17
gene, and P5 through P8 each represent a PCR primer.
[0022] FIG. 7 is a schematic diagram showing the structures of
genetic constructs p2ACt-dAN-gdh and p2AMt-dAN-gdh containing
AN-gdh-17 gene from Aspergillus nidulans connected to 2A11
promoter.
[0023] FIG. 8 shows the cloning strategy of tomato NAD-GDH gene
(T-gdh-4) into Ti plasmid (plG121-Hm). In the figure, 35S
represents CaMV 35S promoter, and Nos represents a
Nos-terminator.
[0024] FIG. 9 is a schematic diagram showing the strategy of
modifying T-gdh-4, a tomato NAD-GDH gene.
[0025] FIG. 10 shows the result of PCR analysis of transformants
into which Aspergillus nidulans AN-gdh-17 gene was introduced,
wherein: [0026] lane 1: .lamda.-HindIII marker; [0027] lanes 2 and
3: untransformed tomatoes (Cont-1 and Cont-2); [0028] lanes 4 to 6:
transformed tomatoes obtained by introducing plasmid (pMAT037)
gene; [0029] lanes 7 to 10: transformed tomatoes (No. 6, No. 8-2,
No. 15 and No. 17) obtained by introducing AN-gdh-17 gene; and
[0030] lane 11: 100 bp marker.
[0031] FIG. 11 shows a result of PCR analysis of transformants
obtained by introducing GDH (T-gdh-4) gene which is tomato NAD-GDH
gene. [0032] lane 1: .lamda.-HindIII marker; [0033] lanes 2 and 3:
untransformed tomatoes; [0034] lanes 4 to 6: transformed tomatoes
obtained by introducing plasmid (plGl21-Hm) gene; [0035] lanes 7 to
10: transformed tomatoes (No. 2, No. 7-2, No. 9-2 and No. 10)
obtained by introducing T-gdh-4 gene; and [0036] lane 11: 100 bp
marker.
[0037] FIG. 12 shows a result of RT-PCR analysis of a transformant
into which Aspergillus nidulans AN-gdh-4 gene was introduced,
[0038] lane 1: 100 bp marker; [0039] lane 2: untransformed
tomatoes; [0040] lanes 3 and 5: transformed tomato No. 6; [0041]
lane 4: transformed tomato No. 15. [0042] The tissues from which
the total RNA was extracted were shown in the parenthesis.
[0043] FIG. 13 shows a result of RT-PCR analysis of a transformant
into which tomato T-gdh-4 gene was introduced, wherein Nos. 2, 7-2,
9-2 and 10 each represent a transformed tomato, and tissues from
which the total RNA was extracted were shown in the
parenthesis.
[0044] FIG. 14 is a graph showing the comparison of amino acid
(glutamic acid--Glu, glutamine--Gln, .gamma.-aminobutyric
acid--GABA and lysine--Lys) content of the transformants (No. 6,
No. 15 and No. 17) into which AN-gdh-17 gene was introduced.
[0045] FIG. 15 shows a result of the comparison of amino acid
(glutamic acid--Glu, glutamine--Gln, .gamma.-aminobutyric
acid--GABA and lysine--Lys) content of the transformants (No. 2,
No. 7-2, No. 9-2, No. 10) into which T-gdh-4 gene was
introduced.
[0046] FIG. 16 shows the result of Southern analysis of transgenic
tomatoes (T.sub.1) into which the AN-gdh-17 gene was introduced.
The samples used were prepared by digesting the total DNA (15
.mu.g) with Bam HI and EcoRI (A) or with Xba I (B). [0047] lane 1:
untransformed tomato, [0048] lane 2: AN-gdh-17 No. 1; [0049] lane
3: AN-gdh-17 No. 3; [0050] lane 4: AN-gdh-17 No. 15; [0051] lane 5:
AN-gdh-17 No. 2.1.
[0052] FIG. 17 shows the result of Southern analysis of transgenic
tomato (T1) into which T-gdh-4 gene was introduced. The samples
used were prepared by digesting the total DNA (15 .mu.g) with Xba I
and Sac I. [0053] lane 1: untransformed tomato; [0054] lane 2:
T-gdh No. 1-2; [0055] lane 3: T-gdh No. 3-3; [0056] lane 4: T-gdh
No. 8-1. [0057] The arrow indicates the location of the band
corresponding to the introduced gene (1.2 kb)
[0058] FIG. 18 shows amino acids contents in fruits of T.sub.1
tomato into which AN-gdh-17 gene had been introduced. An
untransformed tomato was used as a control. Each measurement was
conducted using 3 plants.
[0059] FIG. 19 shows amino acids contents in fruits of T.sub.1
tomato into which T-gdh-4 gene was introduced. An untransformed
tomato was used as a control. Each measurement was conducted using
3 plants.
[0060] FIG. 20 shows a Southern analysis of transgenic potatoes
into which Ct-AN-gdh or Mt-dAN-gdh gene was introduced. Eco RI
digest of the total DNA (15 .mu.g) was used. [0061] lane 1:
untransformed potato-1; [0062] lane 2: untransformed potato-2;
[0063] lanes 3-6: corresponding to a transformed potato into which
each genetic construct was introduced; lane 3: Ct-AN-gdh No. 1,
lane 4: Mt-dAN-gdh No. 2, lane 5: Mt-dAN-gdh No. 5, lane 6:
Mt-dAN-gdh No. 8. [0064] The arrow indicates the location of the
band corresponding to the introduced gene segment (1.5 kb)
[0065] FIG. 21 is a graph showing the contents of Glu in
microtubers of potato transformed with Ct-AN-gdh or Mt-dAN-gdh
gene. An untransformed potato was used as a control.
[0066] FIG. 22 is a graph showing the fresh weight of the above
ground part of the potato into which Mt-dAN-gdh gene was
introduced.
[0067] FIG. 23 is a graph showing the fresh weight of tubers of the
potato into which Mt-dAN-gdh gene was introduced.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0068] The present invention relates to a genetic manipulation of
the nitrogen metabolism in plant. Particularly, the present
invention relates to modifying the expression level of the enzymes
involved in the nitrogen assimilation or nitrogen utilization to
increase free amino acids, particularly glutamic acid which is the
tasty ingredient, in edible parts of useful plants such as fruits,
tubers, roots of root crops or seeds. These enzymes are enhanced in
their expression or modified, or inhibited to produce the pants
having the desired properties.
[0069] The target genes used in the present invention are those
encoding enzymes concerning the assimilation of ammonia to amino
acids. An example of the target genes is glutamate dehydrogenase
(GDH). The expression of this enzyme is enhanced and, in addition,
it is modified (for example, ectopic expression by addition of a
transit sequence) to produce a plant having desired properties.
Particularly, the yielding of potato may be increased by enhancing
or modifying the expression (for example, ectopic expression by the
addition of a transit sequence) of GDH in potato.
[0070] The manipulation may be carried out by transforming a plant
with a nucleic acid construct described herein. The transformed
plants or their descendants express desired, modified enzymes and
they are screened for attaining a change in expression of
corresponding mRNA, change in the assimilation or utilization of
nitrogen and/or increase in free amino acid content of plants.
[0071] In short, the method of the present invention comprises the
following steps, and the transgenic plants of the present invention
are those produced by this method: [0072] a) the step of cloning
the intended gene; [0073] b) the step of recloning the obtained
gene into a suitable vector, if necessary; [0074] c) the step of
introducing the vector into plant cells to obtain a transformant;
and [0075] d) the step of regenerating the obtained transformant to
a plant and cultivating it.
[0076] In one of the embodiments of the present invention, one or
several genes encoding the enzyme(s) for assimilating or utilizing
nitrogen are placed under the control of a strong constitutive
promoter, and over-expressed in the plant bodies. The modification
of the expression may be accomplished by the gene manipulation of
the plants by utilizing at least one of the followings: [0077] a) a
transgene in which a gene sequence encoding an enzyme is operably
connected to a strong constituting promoter, [0078] b) native multi
copy genes, which encodes a desired enzyme, [0079] c) a regulatory
gene for activating the expression of an intended gene for
assimilating or utilizing nitrogen. [0080] d) a native single copy
gene, which is modified in order to increase the expression and
which has a regulatory site, and [0081] e) a transgene which
expresses a variant, modified or chimera enzyme for assimilating or
utilizing nitrogen.
[0082] In another embodiment of the present invention, the
expression pattern of the enzyme for assimilating or utilizing
nitrogen is modified. The expression pattern may be modified by the
gene manipulation of the plants by utilizing at least one of the
followings: [0083] a) a transgene in which a gene sequence encoding
an enzyme is operably connected to a promoter having a desired
expression pattern (such as a promoter having an organ-specific or
development stage-specific expression pattern). [0084] b) a
modification regulatory gene which activates the expression in a
preferred pattern of a gene encoding the enzyme, and [0085] c) a
native single copy gene, which has a regulatory site, modified so
as to express in a preferred pattern.
[0086] In still another embodiment of the present invention, a
modified enzyme or enzyme of a different type is represented in a
pathway of assimilating or utilizing nitrogen. This type of
embodiment involves the production of a genetic construct which can
be expressed in plant cells and which encodes a corresponding
enzyme having a catalytic effect different from a catalytic effect
of the enzyme, which assimilates or utilizes nitrogen in a host
plant and also the gene manipulation with the genetic construct. By
such procedures, plants containing free amino acids in an increased
amount may be obtained.
[0087] For breeding such plants, a conventional method of breeding
new varieties of plants is unsuitable because it requires screening
of large isolated groups and a long time. However, by employing the
methods of the present invention, such a labor becomes unnecessary
and the time can be saved.
[0088] The terms and abbreviations used herein are defined as
follows: [0089] CaMV35S: cauliflower mosaic virus 35S promoter
[0090] NADP-GDH: NADP-dependent glutamate dehydrogenase [0091]
NAD-GDH: NAD-dependent glutamate dehydrogenase [0092] Fused gene
construct: a genetic construct comprising a promoter in which
different genes are connected together (the promoter controls the
transcription of heterologous genes) [0093] Heterologous gene: In a
genetic construct, a heterologous gene means a gene which is
connected with a promoter which is not naturally linked to the
gene. The heterologous gene may be from the organism which provide
the promoter. [0094] GABA: .gamma.-aminobutyric acid.
[0095] The genes of the enzymes usable in the present invention may
be derived from, but not limited to, bacteria, yeasts, alga,
animals and plants. They can be obtained also from various other
sources. The sequences obtained from those sources may be connected
to a suitable promoter which functions in plant cells in such a
manner that the function is not disturbed. The in vitro mutagenesis
or de novo synthesis is also possible in order to enhance the
translation efficiency in the host plants or to change the
catalytic effect of the encoded enzyme. The modification includes
the modification of the residue concerning catalytic functions but
is not limited thereto. The transgene can be modified so as to have
an optimum codon depending on the codon usage of the host or the
organelle to be expressed. If necessary, such a gene sequence may
be connected to a nucleic acid sequence encoding a suitable transit
peptide.
[0096] A preferred modification also includes a construction of
hybrid enzymes. For example, different domains of related enzymes
obtained from the same or different organisms may be combined with
each other to generate an enzyme having a new property.
[0097] In addition, nucleic acid segments capable of hybridizing
with the above described various nucleic acid sequences under
stringent conditions can also be used in the present invention so
far as the desired activity is not lost. Thus, nucleic acid
segments encoding a protein, in which one or more amino acids are
deleted, added or replaced, are also included. The term "stringent
conditions" indicates ordinary conditions well known by those
skilled in the art such as described by Sambrook et al [as
described above (1989)]. Nucleic acid sequences capable of
hybridizing under such conditions will usually have at least 60%,
preferably at least 80% and particularly preferably at least 90% of
homology to each other.
[0098] Various genes are included in the enzyme genes for
assimilating or utilizing nitrogen usable in the present invention.
Glutamate dehydrogenase (GDH) gene is one of examples of preferred
enzymes usable for accumulating glutamic acid. When GDH gene is
used, it is expressed in the sense direction. When GDH gene is
selected, it is preferably expressed as a fusion gene having a
transit peptide at the 5' region. Particularly preferred transit
peptides are a transit peptide for mitochondria and that for
chloroplast.
[0099] A preferred embodiment of the present invention will be
illustrated with reference to an example wherein a tomato plant or
a potato plant is manipulated by a genetic engineering technique
with a recombinant genetic construct encoding NADP-dependant GDH
gene from a fungus (Aspergillus nidulans) [Alastair et al., Mol.
Gen. Genetics, 218, 105-111 (1989)] or tomato NAD-dependant GDH
gene [Purnell et al., Gene 186; 249-254 (1997)] functionally
connected to the cauliflower mosaic virus (CaMV) 35S promoter which
is a powerful constitutive plant promoter. In the lines where GDH
is excessively expressed, free amino acid content in the edible
parts is increased as compared with that of the control
untransformed plant. In particular, glutamic acid content is
increased to 2- to 3-fold.
[0100] The nucleic acid constructs usable in the present invention
can be prepared by the methods well known by those skilled in the
art. For example, the recombinant DNA techniques which can be used
for isolating the components of a construct, determining their
features, handling them, and generating the construct, can be found
in, for example, Sambrook et al., Molecular cloning-Laboratory
manual, the second edition (Cold Spring Harbor Laboratory Press).
When a nucleotide sequence of a desired component is known, it is
advantageous not to isolate it from a biological source but to
synthesize it. In such a case, those skilled in the art may refer
to literatures such as Caruthers et al., Nuc. Acids. Res. Symp.
Ser. 7: 215-233 (1980) and Chow and Kempe, Nuc. Acids. Res. 9:
2807-2817 (1981). In other cases, the desired component may be
advantageously produced by polymerase chain reaction (PCR)
amplification. As for PCR method, those skilled in the art can
refer to Gelfand, "PCR Technique (The Theory and Application of DNA
Amplification)" edited by H. A. Erlich and published by Stockton
Press, N. Y. in 1989 and "Present Protocol in Molecular Biology"
Vol. 2, Chapter 15 edited by Ausubel et al., and published by John
Wiley & Sons in 1988.
[0101] The genetic constructs used in the present invention may
generally contain a suitable promoter which functions in plant
cells, a suitable terminator such as nopaline synthetic enzyme gene
terminator, other elements useful for regulating the expression and
marker genes suitable for selecting the transformant such as
drug-resistant genes, e. g. genes resistant to kanamycin, G 418 or
hygromycin in addition to the intended gene. The promoter contained
in the genetic construct may be a constitutive promoter, an
organ-specific promoter or a developmental stage-specific promoter
and can be suitably selected depending on the host, gene, desired
expression level, organ for the expression, developmental stage,
etc.
[0102] According to the present invention, a plant showing an
overexpression of an enzyme for assimilating or utilizing nitrogen
can be obtained by transforming plant cells with a genetic
construct containing a plant promoter connected to a sequence
encoding a desired enzyme. In a preferred embodiment of the present
invention, related promoters are powerful, organ-unspecific or
developmental stage-unspecific promoters (such as promoters which
strongly express in many or all tissues). An example of such a
powerful constitutive promoters is CaMV35S promoter.
[0103] In another embodiment of the present invention, it is
advantageous in some cases that a plant is manipulated with a
genetic construct in which an organ-specific or growing
stage-specific promoter is linked with a sequence encoding a
desired enzyme. For example, when the expression in a
photosynthetic tissues and organs is intended, a promoter of
ribulose bisphosphate carboxylase (RuBisCO) gene or chloroplast a/b
binding protein (CAB) gene is usable. When the expression in seeds
is intended, promoters of various seed storage protein genes are
usable. When the expression in fruits is intended, a fruit-specific
promoter (such as tomato 2A11) is usable. When the expression in
tubers is intended, a promoter of protein genes stored in tubers
(such as potato patatin) is usable.
[0104] In still another embodiment of the present invention, it may
be advantageous to transform a plant with a genetic construct in
which an inducible promoter connected to a sequence encoding the
desired enzyme. Examples of such promoters are diversified, which
include but are not limited to: heat shock genes, protection
responding genes (such as phenylalanine ammonia lyase gene), wound
responding genes (such as cell wall protein genes rich in
hydroxyproline), chemically inducible genes (such as nitrate
reductase gene and chitinase gene) and dark inducible genes (such
as asparagine synthetase gene (Coruzzi and Tsai, U.S. Pat. No.
5,256,558).
[0105] The recombinant nucleic acid genetic construct of the
present invention may contain a selectable marker for tracing the
transmission of the genetic construct. For example, a genetic
construct transmitted in bacteria preferably contains an
antibiotic-resistant gene such as kanamycin resistant, tetracycline
resistant, streptomycin resistant or chloramphenicol resistant
genes. The suitable vectors to transfer the genetic construct
include plasmids, cosmids, bacteriophages and viruses. In addition,
the recombinant genetic construct may contain a selectable marker
gene or a marker gene which can be screened, which can be expressed
in plants, for isolating, identifying or tracing the plant cells
transformed with the genetic construct. The selective markers
include, but are not limited to, genes which impart resistance to
an antibiotic (such as kanamycin or hygromycin) or resistance to a
herbicide (such as sulfonylurea, phosphinothricin or glyphosate).
The markers which may be screened include, but are not limited to,
genes encoding .beta.-glucuronidase [Jefferson, Plant Mol. Biol.
Rep 5: 387-405 (1987)], genes encoding luciferase [Ow et al.,
Science 234: 856-859 (1986)] and B and Cl gene products controlling
the production of anthocyanin pigment (Goff et al., EMBO J, 9:
2517-2522 (1990)).
[0106] The methods of gene introducing which may be employed in the
present invention are not particularly limited. Any method known in
the art for transferring a gene into plant cells or plant bodies
may be employed. For example, in an embodiment of the present
invention, Agrobacterium may be used for introducing a genetic
construct into a plant. In such a transformation, it is desirable
to use binary Agrobacterium T-DNA vector [Bevan, Nuc. acid Res. 12:
8711-8721 (1984)] and co-culture [Horsch et al, Science, 227:
1229-1231 (1985)]. Agrobacterium transformed system is usually used
for manipulating dicotyledons [Bevans et al., Ann. Rev. Genet., 16:
357-384 (1982); and Rogers et al., Methods Enzymol., 118: 627-641
(1986)]. Agrobacterium transformed system is also usable for
transforming monocotyledons and plant cells [Hernalsteen et al.,
EMBO J., 3: 3039-3041 (1984); Hoykass-Van Slogteren et al., Nature,
311: 763-764 (1984); Grimsley et al., Nature, 325: 167-1679 (1987);
Boulton et al., Plant Mol. Biol., 12: 31-40 (1989); and Gould et
al., Plant Physiol., 95: 426-434 (1991)]. When the Agrobacterium
system is used for the transformation of plants, the recombinant
DNA genetic construct further contains at least right border
sequence of T-DNA region at a position adjacent to DNA sequence to
be introduced into plant cells. In a preferred embodiment, the
sequence to be introduced is inserted between the left and right
T-DNA border sequences. Suitable designs and constructions of
transformed vectors based on T-DNA are well known in the art.
[0107] In another embodiment, various other methods may be employed
for introducing the recombinant nucleic acid genetic construct into
plants or plant cells. An example of other gene introduction method
and transformation method is a protoplast transformation of naked
DNA by calcium, polyethylene glycol (PEG) or electroporation
[Paszkowski et al., EMBO J., 3: 2717-2722 (1984); Potrykus et al.,
Mol. Gen. Genet., 199: 169-177 (1985); Fromm et al., Proc. Nat.
Acad. Sci. USA, 82: 5824-5828 (1985); and Shimamoto et al., Nature,
338: 274-276 (1989)]. According to the present invention, various
plants and plant cells can be manipulated to obtain desired
physiological properties described herein by using the nucleic acid
construct and the transformation method, as described above. The
methods of the present invention are particularly advantageous when
the target product is a monocotyledon or plant cells. In a
preferred embodiment, the target plants and plant cells to be
manipulated include, but are not limited to, tomato, potato, beet,
soybean, Arabidopsis, maize, wheat, rice plant and sugar cane.
[0108] According to the present invention, an intended plant can be
obtained by introducing and manipulating a genetic construct as
disclosed herein into various plant cells including, but are not
limited to, protoplasts, tissue-cultured cells, tissues and organ
explants, pollens, embryos and whole plant bodies. From the plants
manipulated according to the embodiment of the present invention,
the intended transgenic plant is selected or screened by an
approach and method described below. An individual plant body may
be regenerated from the isolated transformant. Methods of
regenerating individual plant bodies from plant cells, tissues or
organs for various species are well known by those skilled in the
art.
[0109] The transformed plant cells, calli, tissues or plants may be
identified and isolated by selecting or screening the characters
encoded by marker genes contained in the genetic construct used for
the transformation. For example, the selection may be conducted by
growing a manipulated plant in a medium containing a repressive
amount of antibiotic or herbicide, to which the introduced genetic
construct can impart the resistance. Further, the transformed plant
cells and plants may be identified by the screening with reference
to the activity of visible marker genes (such as
.beta.-glucuronidase genes, luciferase genes, B genes or Cl genes)
which may be present in the transgenic nucleic acid construct of
the present invention. The methods of the selection and screening
are well known by those skilled in the art.
[0110] Physical methods and biochemical methods can be employed for
identifying plants containing the genetic construct of the present
invention or plant cells transformed with the construct. Examples
of the methods include: [0111] 1) Southern analysis or PCR
amplification for detecting and determining the structure of
recombinant DNA insert; [0112] 2) Northern blotting, S1 RNase
protection, primer elongation PCR amplification or reverse
transcriptase PCR (RT-PCR) amplification for detecting and
determining the RNA transcription product of genetic construct; and
[0113] 3) When the genetic construct is a protein, protein gel
electrophoresis, western blotting, immune precipitation, enzyme
immunoassay, etc. but the methods are not limited to them. These
assay methods are well known by those skilled in the art.
[0114] According to the present invention, the transformed plant
can be screened for an intended physiological change for the
purpose of obtaining the plant having improved component
characters. For example, when the manipulation is conducted for
overexpression of GDH enzyme, the transformed plant can be tested
for the expression of GDH enzyme at a desired level in a desired
tissue or in a desired growing stage. Then the plant having a
desired physiological change, such as overexpression of GDH gene,
may be successively screened with reference to a desired change in
the components.
[0115] According to the present invention, plants manipulated by
modifications in the process for assimilating or utilizing nitrogen
have improved component characteristics. Namely, they may contain a
large amount of free amino acids, particularly glutamic acid,
asparagine, aspartic acid, serine, threonine, alanine and
histidine, especially they may contain glutamic acid in a
particularly large amount, which is a tasty component. The
manipulated plants and plant lines having such improved characters
can be identified by determining free amino acid contents of the
plants. The operation and method of the analysis are well known by
those skilled in the art. Additionally, the yield of potato can be
increased according to the present method. The potato having this
improved property may be identified by simply cultivating potatoes
and determining the number of tubers or the total weight of the
tubers and the like.
[0116] The plants obtained by the present invention have free amino
acid contents higher than those of control plants (untransformed
plants). Untransformed plant as used herein is a plant that is not
yet transformed with a genetic construct which is capable of
expressing GDH. In a preferred embodiment, free amino acid content,
particularly glutamic acid (tasty component) content in edible
parts such as fruits, tubers, roots and seeds of a desired plant is
increased to at least twice as high as that of the parent. The
total amino acid content is also increased to 2 to 4 times as high
as that of the parent. As for amino acids other than glutamic acid,
the increase in amount of particularly aspartic acid, asparagine,
alanine, serine, threonine and histidine is remarkable.
[0117] The potatoes which may be obtained according to the present
invention, are the plant having increased yield compared to a
control plant (untransformed plant). Untransformed plant is a plant
which is not yet transformed with a genetic construct which is
capable of expressing GDH. In a preferred embodiment, the total
weight (g) of the tuber part increases in statically significant
amount, generally at least 1.5-fold, preferably at least 2-fold,
and the number of tubers also increases.
EXAMPLES
[0118] The present invention will be concretely illustrated in
detail by the following Examples relating to the production of
engineered plants for overexpression of NADP-GDH gene or NAD-GDH
gene.
Example 1
Isolation of GDH Gene from Aspergillus nidulans and Tomato, and
Construction of Ti Plasmid
(1) Isolation of NADP-Dependent GDH Gene (AN-gdh-17) from
Aspergillus nidulans and NAD-Dependent GDH Gene (T-gdh-4) from
Tomato
[0119] A. nidulans was plated and cultured on potato dextrose agar
medium at 30.degree. C. overnight. The colonies thus obtained were
further cultured in a dextrose liquid medium for 2 days. Total RNA
was produced from the propagated microbes.
[0120] Tomato seeds surface-sterilized with 70% ethanol (30
seconds) and 2% sodium hypochlorite (15 minutes) were placed on
plant hormone-free MS agar medium [Murashige and Skoog, Physiol.
Plant., 15: 473-479 (1962)], and cultured at 25.degree. C. for one
week while the daylight hours were kept to be 16 hours to obtain
sterile plants. Total RNA was prepared from the roots of the
obtained seedlings.
[0121] As for the total RNA, mRNA was purified with Poly (A) Quick
mRNA Isolation Kit (Stratagene Co.) and then First-Strand cDNA was
produced with First-Strand cDNA Synthesis Kit (Amersham Pharmacia
Biothech Co.). The PCR reaction was conducted with First-Strand
cDNA, thus obtained, as a template. The PCR reaction was conducted
with PCR system 2400 (Perkin Elmer) as follows: 35 cycles under
conditions of 94.degree. C.--3 minutes, 94.degree. C.--45 seconds,
59.degree. C.--30 seconds, 72.degree. C.--90 seconds; and then
72.degree. C.--10 minutes. The primers used are shown in Table 1.
As a result, a band of about 1.4 kbp from A. nidulans and that of
about 1.2 kb from tomato were amplified and they were coincident
with the expected sizes of the intended genes. Obtained PCR
products were cloned with TA-cloning kit (Invitrogen Co.).
[0122] The sequences of 2 clones of plasmids into which the genes
of an intended size from A. nidulans were cloned and also the
sequences of 5 clones of plasmids into which the genes of an
intended size from a tomato root were determined with a sequencer
(377A of ABI Co.), and the homology of them to known NADP-dependent
GDH gene from A. nidulans [Alastair et al., Mol. Gen. Genet. 218;
105-111 (1989)] and GDH gene from tomato [Purnell et al., Gene 186;
249-254 (1997)] was examined.
[0123] The nucleotide sequence of one of two clones derived from A.
nidulans (AN-gdh-17) coincided with known nucleotide sequence of
NADP-GDH gene (FIG. 1-2). However, it was found that in two
splicing sites, one splicing site of about 50 bp remained in the
gene. Because A. nidulans has eucaryote-type splicing recognition
site, the experiment in the subsequent step was conducted with the
remaining splicing site. On the other hand, nucleotide sequences of
2 clones (T-gdh-4 and T-gdh-22) in 5 clones derived form a tomato
root coincided with a known legdh 1 sequence. The nucleotide
sequence of AN-gdh-17 is shown in SEQ ID NO:1, and that of T-gdh-4
is shown in SEQ ID NO:2. TABLE-US-00001 TABLE 1 Primer DNA used for
PCR reaction a. 5' -region -TCT AGA ATG TCT AAC CTT CCC GTT GAG C-
3' -region (28 mer) (SEQ ID:3) 5' -region -GAG CTC TCA CCA CCA GTC
ACC CTG GTC C- 3' -region (28 mer) (SEQ ID:4) b. 5' -region -TCT
AGA ATG AAT GCT TTA GCA GCA ACT- 3' -region (27 mer) (SEQ ID:5) 5'
-region -GAG CTC TTA CGC CTC CCA TCC TCG AAG- 3' -region (27 mer)
(SEQ ID:6) a. NADP-GDH gene specific primer [Alastair et al. Mol.
Gen. Genet., 218; 105-111 (1989), PCR product, about 1.4 kbp] b.
legdh1 specific primer [Purnell et al., Gene 186; 249-254 (1997),
PCR product, about 1.2 kbp]
<Sequence Listing Free Text> [0124] SEQ ID NOs:3,4: NADP-GDH
specific PCR primer [0125] SEQ ID NOs:5,6: legdh1 specific PCR
primer (2) Subcloning of AN-gdh-17 Gene Into Ti-Plasmid
(pMAT037)
[0126] AN-gdh-17 gene cloned into PCR2.1 vector was subcloned into
Ti plasmid (pMAT037) which was a plant transformation vector
[Matsuoka and Nakamura, Proc. Nat. Acad. Sci. USA, 88: 834-838
(1991)]. Because pMAT037 did not have restriction enzyme sites
suitable for the direct insertion, the gene was once ligated into
pUC18 (XbaI, EcoRI site used) to transform E. coli JM109. It was
then ligated with Ti plasmid through PstI site and EcoRI site in
pUC18 to obtain plasmid pAN-gdh-17 (FIG. 3 and Table 2). The
transformation into E. coli DH5 .alpha. was conducted.
Agrobacterium strain EHA101 was transformed with Ti plasmid
pAN-gdh-17 (FIG. 3) in which AN-gdh-17 had been introduced.
Obtained Agrobacterium pAN-gdh-17/EHA101 was used for the infection
of tomatoes.
(3) Construction of pCt-AN-gdh, pCt-dAN-gdh and pMt-dAN-gdh
[0127] NADP-GDH gene from Aspergillus nidurans has originally two
splicing sites. Although these splicing sites should have been
removed by the amplification of GDH gene with cDNA by PCR method,
AN-gdh-17 gene obtained in the experiment still had one splicing
site of about 50 bp remaining therein (FIG. 1, see nucleotide
sequence). Therefore, the remaining nucleotide sequence of about 50
bp was removed by PCR method (Table 2, FIGS. 4-5).
[0128] The strategy for the construction of these genetic
constructs is shown in FIG. 5. In this strategy, DNA segments were
amplified by PCR method by using primer P1 containing 5' terminal
of the cloned gene sequence, primer 2 containing 5' side and 3'
side of the splicing site but free of the splicing site, primer P3
containing 5' side and 3' side of the splicing site but free of the
splicing site, and primer P4 containing 3' terminal of AN-gdh-17
gene and also using AN-gdh-17 gene as the template. Then the size
of the PCR product was confirmed by the electrophoresis and the
product was re-extracted from the gel. The re-extracted PCR
products were mixed together. PCR reaction was conducted again by
using primers P1 and P4. The obtained PCR products were cloned and
sequenced to confirm that the splicing site was accurately
removed.
[0129] The sequences of the above-described P1 to P4 were as
follows: TABLE-US-00002 (SEQ ID:7) P1;
5'-TCTAGAATGTCTAACCTTCCCGTTGAGC-3' (SEQ ID:8) P2;
5'-CACCCATGTTTAGTCCTGTGAGAG-3' (SEQ ID:9) P3;
5'-CTCTGACAGGACTAAACATGGGTG-3' (SEQ ID:10) P4;
5'-GAGCTCTCACCACCAGTCACCCTGGTCC-3'
[0130] Thus, dAN-gdh-17 gene from which the splicing site had been
removed was obtained (Table 2, FIGS. 4-5).
[0131] To make the action of the introduced gene more functional at
a suitable position, the transit peptide sequence for mitochondria
or chloroplast was connected to the upstream of the initiation
codon of AN-gdh-17 gene or dAN-gdh-17 gene (Table 2, FIG. 4). As
for the peptide sequences used, a nucleotide sequence (about 70 bp)
connected to tomato GDH gene was used as the transit peptide
sequence for mitochondria, and a nucleotide sequence (about 120 bp)
connected to the small subunit gene of tomato RuBisCO was used as
the transit peptide sequence for chloroplast. These genes were
connected to AN-gdh-17 gene or dAN-gdh-17 gene by PCR method. The
transit peptide sequence for mitochondria was obtained by using two
primers(5'-GGATCCATGAATGCTTTAGCAGCAAC-3': sequence SEQ IDNO:11, and
5'-TCTAGATAAACCAAGAAGCCTAGCTG-3': sequence SEQ IDNO:12) by PCR. The
transit peptide sequence for chloroplast was obtained by using two
primers (5'-CTGCAGATGGCTTCCTCAATTGTCTCATCG-3': sequence SEQ ID
NO:13, and 5'-TCTAGAGCATCTAACGCGTCCACCATTGCT-3': sequence SEQ ID
NO:14) by PCR.
[0132] These transit peptide sequences were linked with AN-gdh-17
gene or dAN-gdh-17 gene as shown in FIG. 6. Namely, the respective
DNA segments were amplified by using primer P5 corresponding to 5'
side of the transit peptide, primer P6 containing the sequence of
3' terminal of the transit peptide and the sequence at 5' terminal
of AN-gdh-17 gene or dAN-gdh-17 gene, primer P7 containing
sequences of 3' terminal of the transit peptide and 5' terminal of
AN-gdh-17 gene or dAN-gdh-17 gene, and primer P8 containing
sequences of 3' terminal of AN-gdh-17 gene or dAN-gdh-17 gene. Then
the size of the PCR product was confirmed by the electrophoresis.
After the extraction from the gel, the extracted segments were
mixed together and again subjected to PCR with primers P5 and p8.
The amplified segments were cloned and then sequenced to confirm
that the nucleotide sequence of the transit peptide was correctly
connected to AN-gdh-17 gene or dAN-gdh-17 gene (FIG. 6).
[0133] The sequences of P5 to P8 are as shown below.
[0134] The primer used for linking with the transit peptide
sequence for mitochondria: TABLE-US-00003 (SEQ ID:15) P5;
5'-TCTAGAATGAATGCTTTAGGAGCAAC-3' (SEQ ID:16) P6;
5'-GGGAAGGTTAGACATTAAACCAAGAAGCCT-3' (SEQ ID:17) P7;
5'-AGGCTTCTTGGTTTAATGTCTAACCTTCCC-3' (SEQ ID:18) P8;
5'-GAGCTCTTACGCCTCCCATCCTCGAA-3'
[0135] The primer used for linking with the transit peptide
sequence for chloroplast: TABLE-US-00004 (SEQ ID:19) P5;
5'-CTGCAGATGGCTTCCTCAATTGTCTCATCG-3' (SEQ ID:20) P6;
5'-AAGGTTAGACATGCATCTACCGGG-3' (SEQ ID:21) P7;
5'-GGCGTTAGATGCATGTGTAACCTT-3' (SEQ ID:22) P8;
5'-GAGCTCTTACGCCTCCCATCCTCGAA-3'
[0136] AN-gdh-17 gene was introduced into the sense direction in
the multicloning site of Ti plasmid pMAT037 (FIG. 3). In the
construction of pCt-AN-gdh, pCt-dAN-gdh and pMt-dAN-gdh, the
cloning was conducted with Ti plasmid plG121-Hm and the gene was
introduced in order to compare the effect obtained by using CaMV35S
promoter with that obtained by using fruit-specific promoter gene
(2A11) which will be described below (Table 2, FIG. 4).
<Sequence Listing Free Text>
[0137] SEQ ID NOs:7-10: PCR primer for removing splicing region
[0138] SEQ ID NOs:11, 12: PCR primer for amplifying the sequence
encoding the mitochondria transit peptide [0139] SEQ ID NOs:13, 14:
PCR primer for amplifying the sequence encoding the chloroplast
transit peptide [0140] SEQ ID NOs:15-18: PCR primer for producing
the sequence encoding the mitochondria transit peptide-GDH [0141]
SEQ ID NOs:19-22: PCR primer for producing the sequence encoding
the chloroplast transit peptide-GDH (4) Genetic Construction with
Fruit-Specific Promoter (2A11 Promoter)
[0142] Fruit-specific expression promoter (2A11) was obtained by
PCR method by using total DNA obtained from tomato seedlings as the
template. In the primers (SEQ ID NOs:23 and 24) used, the sequences
of restriction enzyme sites HindIII and XbaI used for the
introduction into Ti plasmid were designed respectively.
[0143] The sequences of the primers were as follows: TABLE-US-00005
(SEQ ID NO:23) 5'-AAGCTTATATAACCCAAAATATACTA-3' (SEQ ID NO:24)
5'-TCTAGAGGTACCATTAATTGCTAATT-3'
[0144] After cloning the obtained PCR product with TA cloning kit,
the nucleotide sequence thereof was confirmed by the sequence
analysis. Obtained 2A11 promoter was replaced with CaMV35S promoter
before GUS gene of Ti plasmid plG121-Hm by using the
above-described restriction enzymes, HindIII and XbaI. Then GUS
part was replaced with Ct-dAN-gdh gene or Mt-dAN-gdh gene. The
process for the replacement with Ct-dAN-gdh gene or Mt-dAN-gdh gene
was the same as that employed for CaMV35S promoter. From them,
plasmids p2Act-dAN-gdh and p2AMt-dAN-gdh were obtained (FIG.
7).
<Sequence Listing Free Text>
[0145] SEQ ID NOs:23, 24: PCR primer for amplifying 2A11 promoter
sequence
[0146] Important structures of plasmids produced as described above
are summarized in following Table 2. TABLE-US-00006 TABLE 2
NADP-GDH construct which was introduced into transgenic tomato.
Gene body (NADP-GDH) Splicing region Transit peptide Promoter.
Contain Delete No Chl.*.sup.1 Mit.*.sup.2 35S 2A11 pAN-gdh-17
.largecircle. .largecircle. .largecircle. (FIG. 3) pCt-AN-gdh
.largecircle. .largecircle. .largecircle. (FIG. 4) pCt-dAN-gdh
.largecircle. .largecircle. .largecircle. (FIG. 4) pMt-dAN-gdh
.largecircle. .largecircle. .largecircle. (FIG. 4) p2ACt-dAN-
.largecircle. .largecircle. .largecircle. gdh (FIG. 7) p2AMt-dAN-
.largecircle. .largecircle. .largecircle. gdh (FIG. 7) Chl.*.sup.1:
Chloroplast, Mit.*.sup.2: Mitochondria
(5) Subcloning of T-gdh-4 Gene Into Ti Plasmid (plG121-Hm)
[0147] Cloned tomato GDH gene (T-gdh-4) was introduced into
Ti-plasmid (pIG121-Hm) to obtain plasmid pT-gdh-4. In the
introduction, XbaI site and SacI site previously provided in the
primer used in the isolation step were used. Ti plasmid containing
T-gdh-4 gene (FIG. 8) was used to transform Agrobacterium strain
EHA101.
(6) Subcloning of Tomato GDH Gene Variants Into Ti Plasmid
(plG121-Hm)
[0148] Lysine at position 90 in tomato NAD-GDH which is the
glutamic acid binding site was replaced with alanine, and the
effect obtained by the replacement was examined (FIG. 9). This
modification was conducted for the purpose of inhibiting the
binding with glutamic acid, because GDH gene of higher plants is
inclined to reduce the amount of glutamic acid depending on ammonia
ion concentration and nutrition conditions. The replacement of one
amino acid was conducted by changing AAG encoding Lys to GCG
encoding Ala by site-directed mutagenesis using PCR. The altered
gene thus obtained was named "Td-gdh". Then Td-gdh gene sequence
was introduced into Ti plasmid to obtain plasmid pTd-gdh (FIG. 9).
The strategy is the same as that for T-gdh-4.
Example 2
Production of Tomato Transformants and Analysis of Them
1) Production of Tomato Transformants
[0149] Tomato (cultivar, minitomato, Fukukaen Seeds Co.) seeds were
surface-sterilized with 70% ethanol (30 seconds) and 2% sodium
hypochlorite (15 minutes), and then placed on a plant hormone-free
MS agar medium. The seeds were cultured at 25.degree. C. for one
week while the daylight hours were kept to be 16 hours. The
cotyledons were taken from the obtained sterile seedlings and then
placed on an MS agar medium containing 2 mg/l of Zeatin and 0.1
mg/l of Indoleacetic acid (regeneration medium in 9 cm Petri dish),
and cultured under the same conditions as those described above for
2 days. Agrobacterium (EHA 101) containing thus constructed gene
was cultured in YEP medium (Table 3) overnight and used for the
infection. The cotyledons cultured for two days were collected in a
sterilized Petri dish, and the Agrobacterium suspension was added
to them to cause the infection. Superfluous Agrobacterium
suspension was removed from the cotyledons by using a sterilized
filter paper. After further removing superfluous Agrobacterium
suspension, a sterilized filter paper was then placed on the medium
in the above-described Petri dish in order to prevent rapid
propagation of Agrobacterium. The infected cotyledons were placed
thereon and co-cultured for 24 hours.
[0150] The cotyledons were transferred to MS regeneration medium
(selecting medium) containing 50 mg/l of kanamycin and 500 mg/l of
Claforan to select transformants. The regenerated shoots were
transferred into a new selecting medium to conduct further
selection. Well-grown green shoots were cut at the stems and
transferred into a plant hormone-free MS medium (rooting medium in
a test tube). The rooted, regenerated plant was acclimated to the
soil. TABLE-US-00007 TABLE 3 Composition of YEP medium (1 liter)
Bactotrypton 10 g Yeast extract 10 g glucose 1 g
(2) Confirmation of the Introduced Gene
[0151] Total DNA was extracted by method of Honda et al. [Honda and
Hirai, Jpn. J. Breed 40, 339-348 (1990)] from 4 selected individual
plants obtained by the infection with Agrobacterium containing
AN-gdh-17 gene, 4 selected individuals obtained by the infection
with Agrobacterium containing T-gdh-4 gene, 3 plant individuals
obtained by the infection with Agrobacterium (only Ti plasmid) free
of the intended gene, and 2 plant individuals obtained from the
cotyledons by the direct regeneration without using Agrobacterium.
DNA thus extracted was purified by RNase treatment,
phenol/chloroform treatment and PEG precipitation. The purified
product was diluted to 0.01 .mu.g/.mu.l and used as a template for
PCR. PCR reaction was conducted with primers 9 and 10 which amplify
the region from Nos-Promoter to NPTII (PCR products, 1.0 kbp). The
reaction conditions were as follows: 35 cycles under conditions of
94.degree. C.--1 minute, 55.degree. C.--1 minute and 72.degree.
C.--2 minutes. The PCR product was treated by the electrophoresis
with 1% agarose gel and then stained with ethidium bromide (FIGS.
10 and 11).
[0152] The primers used were as follows: TABLE-US-00008 (SEQ ID
NO:25) P9: 5'-CCCCTCGGTATCCAATTAGAG-3' (SEQ ID NO:26) P10:
5'-CGGGGGGTGGGCGAAGAACTCCAG-3'
[0153] A band of an intended size (1.0 kb) was observed in four
lines infected with AN-gdh-17 gene and four lines infected with
T-gdh-4 gene but not in two untransformed lines. From these
results, it was confirmed that the gene had been introduced into
four lines infected with Ti-plasmid containing AN-gdh-17 gene and
four lines infected with Ti-plasmid containing T-gdh-4 gene.
<Sequence Listing Free Text>
[0154] SEQ ID NO:25, 26: PCR primer for amplifying
Nos-Promoter-NTPII region (3) Confirmation of Expression of
Introduced Gene
[0155] Then, the expression of the introduced gene was confirmed by
RT-PCR by using transformed tomatoes in which the introduction of
the intended gene had been confirmed. The total RNA was extracted
from leaves and fruits of the lines infected with Agrobacterium
containing AN-gdh-17 or T-gdh-4 gene and in which the introduced
genes were confirmed by PCR analysis, and the first strands cDNA
were prepared after RNase treatment. Then PCR was conducted with
primers (SEQ ID NOs:3 and 4, and SEQ ID NOs:5 and 6), which was
used in the isolation of genes, using the first-strand cDNA as the
template. The reaction conditions were as follows: 30 cycles under
conditions of 94.degree. C.--1 minute, 55.degree. C.--1 minute and
72.degree. C.--2 minutes. As a result, the introduced gene was
confirmed to be expressed in all the leaves and fruits (FIGS. 12
and 13).
(4) Extraction and Quantitative Determination of Free Amino
Acids
[0156] Fruits of the acclimated tomato transformant were harvested
6 weeks after the blossom, and stored at -80.degree. C. Each fruit
was cut into about 6 pieces, weighed, placed in a mortar, frozen
with liquid nitrogen and ground. 3 ml of 80% ethanol was added
thereto, and the obtained mixture was further thoroughly ground,
transferred into a centrifugal tube and incubated at 80.degree. C.
for 20 minutes.
[0157] After the centrifugation at 10,000 rpm for 20 minutes, the
supernatant was transferred into another tube. 2 ml of 80% ethanol
was added to the remaining pellets, and the pellets were ground
again in the mortar and then incubated at 80.degree. C. for 20
minutes. After the centrifugation, the supernatant was transferred
into the tube containing the prior supernatant to obtain a mixture.
The total amount of the mixture was adjusted to 5 ml with 80%
ethanol. After thorough mixing, 200 .mu.l of the mixture was taken,
dried and dissolved in 0.02 N hydrochloric acid. After the
filtration through a 0.45 .mu.m filter, the sample for the analysis
was obtained. The amino acid analysis was conducted with Hitachi
high-speed amino acid analyzer (L-8800).
[0158] The amino acid analysis of the fruits (red) of a strain
having AN-gdh-17 gene introduced therein was conducted 6 weeks
after the bloom. The results of the analysis are shown in Table 4
together with the results of the analysis of untransformed plants
(control plants). In the lines wherein glutamic acid content was
remarkably increased, glutamic acid content was increased 1.75-fold
(No. 6), 2.54-fold (No. 15) and 2.48-fold (No. 17) (FIG. 14). Amino
acids other than glutamic acid, such as asparagine, aspartic acid,
alanine, serine, threonine and histidine, were also increased in
amount. TABLE-US-00009 TABLE 4 Amino acid content of transformed
tomato (T.sub.0 generation) into which AN-gdh-17 gene was
introduced Asp Thr Ser Asn Glu Gln Gly Ala GABA His Total
Untransformed tomato Control-1 1.31 0.10 0.14 0.26 8.88 0.68 0.02
0.30 0.45 0.13 12.77 Control-2 1.04 0.07 0.14 0.21 8.48 0.39 0.02
0.22 0.45 0.13 11.90 Plasmid (pMAT037) alone pMAT-1 1.33 0.14 0.32
0.23 6.00 0.70 0.04 0.66 0.14 0.17 10.31 pMAT-2 2.00 0.20 0.37 0.23
11.18 0.50 0.06 0.83 0.62 0.25 17.09 pMAT-3 1.28 0.22 0.44 0.37
3.27 0.85 0.14 0.13 0.22 0.25 12.22 AN-gdh-17 gene introduced
transformant No. 6 1.37 0.29 0.70 0.42 15.16 0.65 0.15 1.59 0.46
0.39 22.86 No. 8-2 2.66 0.18 0.51 0.27 13.97 0.65 0.07 1.19 0.27
0.32 20.82 No. 15 5.51 0.43 0.94 1.18 22.08 4.29 0.16 2.53 1.98
0.53 41.42 No. 17 3.19 0.24 0.69 0.38 21.55 0.67 0.12 1.75 0.82
0.37 30.70 (.mu.mol/g F.W.)
[0159] The amino acid analysis of the 4 strains containing tomato
T-gdh-4 gene introduced therein was conducted by using the fruits
taken 6 weeks after the blooming (Table 5). In the plant lines
wherein the remarkable increase in glutamic acid content was
observed, glutamic acid content was increased to 2.28 times (No.
2), 3.52 times (No. 7-2), 2.74 times (No. 9-2) and 2.53 times 10
(No. 10) (FIG. 15). In the plant lines of a high glutamic acid
content, amino acids other than glutamic acid, such as aspartic
acid, asparagine, threonine, serine, alanine and histidine were
also increased in amount. The total amino acid content was
increased to 4 times (No. 7-2). The results are summarized in Table
5. TABLE-US-00010 TABLE 5 Amino acid content of transformed tomato
(T.sub.0 generation) into which T-gdh-4 gene was introduced Asp Thr
Ser Asn Glu Gln Gly Ala GABA His Total Untransformed tomato
Control-1 1.31 0.10 0.14 0.26 8.88 0.68 0.02 0.30 0.45 0.13 12.77
control-2 1.04 0.07 0.14 0.21 8.48 0.39 0.02 0.22 0.45 0.13 11.90
Plasmid (plG121-Hm) alone plG-1 2.13 0.20 0.37 0.36 9.40 1.07 0.07
1.02 0.33 0.26 16.18 plG-2 1.03 0.12 0.27 0.16 6.59 0.30 0.07 0.68
0.23 0.15 10.10 plG-3 0.67 0.07 0.21 0.20 3.64 0.15 0.03 0.35 0.14
0.26 5.89 T-gdh-4 gene introduced transformant No. 2 4.01 0.27 0.49
0.64 19.76 0.75 0.08 1.09 0.59 0.37 28.93 No. 7-2 6.21 0.55 0.56
0.91 30.56 4.71 0.08 0.80 3.20 0.74 50.15 No. 9-2 4.27 0.69 1.15
1.14 23.81 3.97 0.23 2.25 1.04 0.70 42.17 No. 10 6.78 0.37 0.87
0.62 21.96 1.21 0.15 1.83 2.41 0.63 38.03 (.mu.mol/g F.W.)
Example 3
Analysis of Subsequent Generation (T.sub.1) of Tomato
Transformant
1) Selection of T.sub.1 Generation
[0160] Seeds of transformed tomato (T.sub.0 generation) obtained in
Example 2 and several lines of the transformants (T.sub.0
generation) obtained by similar procedure were surface-sterilized
with 80% ethanol for 30 seconds and 2% 10 sodium hypochlorite for
15 minutes, and then planted in MS agar medium containing 350 mg/l
of kanamycin under sterile conditions. One month later, well-grown
plants were selected to obtain the selected plant bodies from No.
1, 3, 15 and 2.1 in AN-gdh-17 transgenic lines and from No. 1, 3,
and 8 in T-gdh-4 transgenic lines. The plants were cultured in an
outdoor closed system greenhouse in order to increase the number of
fruits per plant. To make the nutrition conditions uniform, no
additional fertilizer was given after the transplantation into 1 kg
of culture soil (Power soil; Sakata no Tane) during the acclimation
to the soil. In the following analysis, leaves were not picked in
order to uniform the assimilation, and the lateral buds were
cultivated under uniform conditions. The leaf tissues of the thus
obtained were used.
(2) Confirmation of the Introduced Gene by Southern Analysis
[0161] Total DNA was extracted from leaf tissues of lateral buds of
the acclimated plant [Honda and Hirai, Jpn. J. Breed 40, 339-348
(1990)]. 15 .mu.g of DNA was treated with the combination of
restriction enzymes BamHI and EcoRI and also reacted with XbaI.
After electrophoresis, it was transferred onto a nylon membrane.
The obtained product was then subjected to Southern hybridization
with a DIG-Labeling and Detection Kit (Roche Molecular
Biochemicals) using AN-gdh-17 gene or T-gdh-4 gene as the
probes.
[0162] Southern hybridization was conducted with AN-gdh-17 gene as
the probe. As a result, bands of intended size (1.8 kb, 0.8 kb)
were observed in No. 1, No. 3, No. 15 and No. 2.1 in AN-gdh-17 gene
transgenic lines and the introduction of the gene was confirmed
(FIG. 16). In the same manner, Southern hybridization was conducted
with T-gdh-4 gene as the probe. A band (1.2 kbp) of a size equal to
that of T-gdh-4 gene was confirmed. In several plants, a band was
observed around the location of 20 kbp, which was considered as the
endogenous GDH gene (FIG. 17).
3) Determination of Activity of NADP-GDH and NAD-GDH
[0163] Leaf tissue (0.2 g) of lateral buds of transformed tomato
(T.sub.1) was frozen in liquid nitrogen, and then crushed in a
mortar. 5-fold weight of an extract buffer [200 mM Tris (pH 8.0),
14 mM .beta.-mercaptoethanol, 10 mM L-cysteine-HCl, 0.5 mM PMSF,
0.5% Triton X-100] was added. The obtained mixture was transferred
into a centrifugal tube and centrifuged at 12,000 rpm at 4.degree.
C. for 10 minutes. The supernatant was ultrafiltrated (Millipore,
ultrafree 0.5 filter unit, Biomax-10) and washed with the
extraction buffer three times.
[0164] The extracted enzyme was mixed with a reaction mixture [100
mM Tris (pH 8.0), 20 mM 2-.alpha.-ketoglutarate, 1.0 mM CaCl.sub.2,
0.2 mM NADPH (for NADP-GDH activity determination) or 0.2 mM NADH
(for NAD-GDH activity determination), 200 mM ammonium chloride],
and the reaction was carried out at room temperature. The reduction
in the absorbance at 340 nm was determined.
[0165] NADP-GDH activity was determined by using leaf tissue of
transformed tomato (T.sub.1) containing AN-gdh-17 gene introduced
therein. The activity of the transformant could be determined to be
230 to 400 nmol/(min.mg protein), while no activity of the
untransformed product was recognized (Table 6). NAD-GDH activity of
the line containing T-gdh-4 gene introduced therein was increased
2-fold or more compared to that of the non-transformant (Table 7).
TABLE-US-00011 TABLE 6 NADP-GDH activity of transformed tomato with
AN-gdh-17 gene. Activity of NADP-GDH Lines (nmol/(min mg protein))
Untransformed tomato 0 Transformed tomato AN-gdh-17 No. 1-1 400
AN-gdh-17 No. 3-1 390 AN-gdh-17 No. 15-1 380 AN-gdh-17 No. 2.1-1
230
[0166] TABLE-US-00012 TABLE 7 NAD-GDH activity of transformed
tomato with T-gdh-4 gene Activity of NAD-GDH Lines (nmol/(min mg
protein)) Untransformed tomato 80 Transformed tomato T-gdh No. 1-2
180 T-gdh No. 3-1 160 T-gdh No. 8-1 260
4) Determination of Amino Acid Content in Fruits
[0167] Three fruits taken in the 6.sup.th week after blossoming of
the first fruit cluster were used for the analysis. 3 parts by
weight of 80% ethanol heated to 80.degree. C. was added to 1 part
by weight of the fruits. The obtained mixture was ground in a
mortar and then heated again to 80.degree. C. for 20 minutes. After
centrifugation at 7,000 rpm, the obtained supernatant was
recovered. After the addition of 80% ethanol, the obtained mixture
was heated to 80.degree. C. The ethanol extraction was conducted
three times, and the obtained extracts were combined together and
then 80% ethanol was added thereto to make the total amount 100 ml.
After thoroughly mixing, 200 .mu.l of the extract was taken in an
Eppendrof tube, dried and then dissolved in 200 .mu.l of sterilized
water. 200 .mu.l of ethyl ether was added to the obtained solution,
and they were mixed together and then centrifuged at 12,000 rpm.
The ether layer was removed. The aqueous layer was dried again and
dissolved in 200 .mu.l of 0.02 N HCl. The resultant solution was
filtered through a 0.45 .mu.m filter, and the filtrate was taken as
a sample and analyzed with Hitachi high-speed amino acid analyzer
(L-8800).
[0168] The results were shown by the average of three fruits.
Glutamic acid contents of AN1-1-2 and AN1-1-3 from AN-gdh-17 No. 1
line were increased to 2.1 times and 2.8 times as high as that of
the untransformed fruit, respectively. Also, glutamic acid contents
of AN3-1-2 and AN3-1-3 from AN-gdh-17 No. 3 line were increased to
2.8 times and 2.5 times as high as that of the untransformed fruit.
Further, glutamic acid contents of AN15-1 from AN-gdh-17 No. 15
line and AN2.1-1-1 from AN-gdh-17 No. 2.1 line were also increased
to 2.1 times and 1.9 times, respectively (Table 8, FIG. 18). The
similar tendency was observed also in the subsequent generation of
No. 15 line whose glutamic acid content was high in the transformed
generation (T.sub.0). TABLE-US-00013 TABLE 8 Amino acid contents in
fruits of the progenies (T.sub.1) of AN-gdh-17 gene introduced
tomato transformants Asp Thr Ser Asn Glu Control 1.09 .+-. 0.48
0.10 .+-. 0.03 0.22 .+-. 0.05 0.18 .+-. 0.09 6.42 .+-. 1.16 AN-GDH
1-1-2 2.50 .+-. 0.78 0.19 .+-. 0.02 0.41 .+-. 0.02 0.38 .+-. 0.04
13.71 .+-. 2.55 AN-GDH 1-1-3 4.17 .+-. 0.15 0.42 .+-. 0.06 0.86
.+-. 0.09 0.63 .+-. 0.13 18.42 .+-. 0.99 AN-GDH 3-1-2 4.18 .+-.
0.96 0.30 .+-. 0.07 0.66 .+-. 0.19 0.50 .+-. 0.18 18.39 .+-. 2.74
AN-GDH 3-1-3 3.37 .+-. 0.89 0.33 .+-. 0.05 0.71 .+-. 0.12 0.57 .+-.
0.07 16.25 .+-. 0.73 AN-GDH 15-1 2.18 .+-. 0.16 0.15 .+-. 0.02 0.36
.+-. 0.02 0.29 .+-. 0.01 13.95 .+-. 0.28 AN-GDH 2.1-1-1 2.33 .+-.
0.38 0.20 .+-. 0.04 0.46 .+-. 0.17 0.23 .+-. 0.06 12.43 .+-. 0.77
Gln Ala His GABA Total Control 0.44 .+-. 0.33 0.29 .+-. 0.11 0.13
.+-. 0.04 0.96 .+-. 0.11 10.36 .+-. 4.24 AN-GDH 1-1-2 0.45 .+-.
0.11 0.62 .+-. 0.16 0.23 .+-. 0.02 2.86 .+-. 0.58 22.18 .+-. 3.71
AN-GDH 1-1-3 1.50 .+-. 1.20 1.54 .+-. 0.15 0.44 .+-. 0.02 5.67 .+-.
0.18 35.46 .+-. 2.88 AN-GDH 3-1-2 1.05 .+-. 0.91 1.25 .+-. 0.84
0.39 .+-. 0.08 3.33 .+-. 0.97 31.76 .+-. 4.76 AN-GDH 3-1-3 1.49
.+-. 1.02 1.18 .+-. 0.48 0.39 .+-. 0.06 2.90 .+-. 0.40 28.68 .+-.
1.22 AN-GDH 15-1 0.27 .+-. 0.10 0.73 .+-. 0.13 0.23 .+-. 0.10 1.09
.+-. 0.13 19.97 .+-. 0.25 AN-GDH 2.1-1-1 0.19 .+-. 0.03 0.91 .+-.
0.47 0.24 .+-. 0.10 1.84 .+-. 0.09 19.59 .+-. 2.29 (.mu.mol/g.F.W.)
(n = 3)
[0169] In T-gdh-4 transgenic lines, glutamic acid content of
T-gdh1-2, 3-1 and 8-1, which were the subsequent generations of
examined T-gdh-4, Nos. 1, 3 and 8 lines, were increased to 2.3
times, 2.1 times and 2.4 times, respectively, as high as that of
the untransformed fruit (Table 9, FIG. 19). As for amino acids
other than glutamic acid, a remarkable increase in aspartic acid
content, glutamine content and .gamma.-amino butyric acid content
was observed. As a result, the total free amino acid content was
also increased 2- to 3-fold as compared to the untransformed plant.
TABLE-US-00014 TABLE 9 Amino acids contents in fruits of the
progenies (T.sub.1) of T-gdh-4 gene introduced tomato transformants
Asp Thr Ser Asn Glu Control 1.09 .+-. 0.48 0.10 .+-. 0.03 0.22 .+-.
0.05 0.18 .+-. 0.09 6.42 .+-. 1.16 T-gdh 1-2 2.61 .+-. 0.51 0.28
.+-. 0.05 0.56 .+-. 0.12 0.48 .+-. 0.02 14.74 .+-. 3.12 T-gdh 3-1
2.41 .+-. 0.51 0.20 .+-. 0.04 0.44 .+-. 0.13 0.30 .+-. 0.01 13.81
.+-. 2.64 T-gdh 8-1 2.85 .+-. 0.60 0.22 .+-. 0.05 0.46 .+-. 0.11
0.31 .+-. 0.07 15.70 .+-. 3.47 Gln Ala His GABA Total Control 0.44
.+-. 0.33 0.29 .+-. 0.11 0.13 .+-. 0.04 0.96 .+-. 0.11 10.36 .+-.
4.24 T-gdh 1-2 0.79 .+-. 0.65 0.91 .+-. 0.56 0.29 .+-. 0.17 1.31
.+-. 0.96 23.29 .+-. 3.88 T-gdh 3-1 0.43 .+-. 0.37 0.70 .+-. 0.35
0.27 .+-. 0.06 1.53 .+-. 0.48 21.09 .+-. 2.80 T-gdh 8-1 0.46 .+-.
0.07 0.69 .+-. 0.05 0.23 .+-. 0.18 2.24 .+-. 0.85 24.22 .+-. 5.19
(.mu.mol/g.F.W.) (n = 3)
Example 4
Production and Analysis of Potato Transformants
(1) Production of Transformants
[0170] Sterile potatoes were obtained by shoot apex culture. The
materials were propagated by shoot apex subculture. The shoot
apexes was placed in a liquid culture medium (10 ml) prepared by
adding 2% sucrose to MS medium to induce the rooting. After
completion of the rooting, 10 ml of MS liquid medium containing 16%
sucrose was added to the medium, and dark culture was conducted to
induce the formation of microtubers. The 6 to 8 week old
microtubers were cut to form disc-shaped pieces. After peeling, the
pieces were infected with an Agrobacterium suspension (Ti-plasmid.
pMt-dAN-gdh or pCt-AN-gdh) cultured at 28.degree. C. overnight. A
sterilized filter paper was placed on MS agar medium (MS medium,
2.0 mg/l Zeatin, 0.1 mg/l indole acetic acid, 0.3% gelrite), the
pieces were placed thereon and co-cultured at 25.degree. C. for 2
days while the daylight hours were kept to be 16 hours. The culture
was then transferred to a selection medium (MS medium, 2.0 mg/l
Zeatin, 0.1 ml/l indole acetic acid, 0.3% gelrite, 50 mg/l
kanamycin and 500 mg/l claforan) and cultured under the same
conditions as those described above. The discs were transferred
into the fresh screening medium every week, and the differentiated
shoots were transferred into a plant hormone-free selection medium
to induce the rooting. After infection with Agrobacterium harboring
Ti-plasmid pMt-dAN-gdh or pCt-AN-gdh and selection on the medium
containing 50 mg/l kanamycin, 4 lines, Mt-dAN-gdh No. 2, 5, 8 and
Ct-AN-gdh No. 1, were obtained.
2) Confirmation of the Introduced Gene by Southern Analysis
[0171] Total DNA was extracted from leaf tissues of the acclimated
plant [Honda and Hirai, Jpn. J. Breed 40, 339-348 (1990)]. 15 .mu.g
of DNA was treated with restriction enzyme EcoRI. After
electrophoresis, it was transferred on a nylon membrane. Southern
hybridization was carried out using DIG-Labeling and Detection Kit
(Roche Molecular Biochemicals). AN-gdh-17 gene was used as the
probe.
[0172] As a result, the band of the intended size (about 1.5 kb)
was confirmed in all 4 lines (FIG. 20), which suggested that gdh
gene connected to the transit peptide was introduced therein.
3) Determination of Activity of NADP-GDH
[0173] Leaf tissues (about 0.1 g) of transformed tomato were frozen
in liquid nitrogen, and then crushed in a mortar. 5-fold weight of
the extract buffer [200 mM Tris (pH 8.0), 14 mM
.beta.-mercaptoethanol, 10 mM L-cysteine-HCl, 0.5 mM PMSF, 0.5%
Triton X-100] was added. The obtained mixture was transferred into
a centrifugal tube and centrifuged at 12,000 rpm for 10 minutes.
The supernatant was ultrafiltrated (Millipore, ultrafree 0.5 filter
unit, Biomax-10) and washed with the extract buffer three times.
The extracted enzyme was mixed with a reaction solution [100 mM
Tris (pH 8.0), 20 mM 2-.alpha.-ketoglutarate, 1.0 mM CaCl.sub.2,
0.2 mM NADPH, 200 mM ammonium chloride], and the reaction was
carried out at room temperature. The reduction in the absorbance at
340 nm was determined.
[0174] NADP-GDH activity was determined by using the leaf tissues
of the transformed potatoes in which the introduced gene could be
confirmed by Southern analysis, and the untransformed potatoes. As
a result, the activity of the transformants could be determined to
be 150 to 300 nmol/l(min.mg protein), while no activity of the
untransformed product was recognized (Table 10). Mt-dAN-gdh lines
showed higher activity than Ct-AN-gdh lines. TABLE-US-00015 TABLE
10 NADP-GDH activity of transgenic potato in which Mt-dAN-gdh or
Ct- AN-gdh gene was introduced Activity of NADP-GDH Line (nmol/(min
mg protein)) Untransformed potato 0 Transformed potato Mt-dAN-gdh
No. 2 290 Mt-dAN-gdh No. 5 300 Mt-dAN-gdh No. 8 260 Ct-AN-gdh No. 1
150
4) Determination of Amino Acid Content in Microtubers
[0175] The shoot apexes of 4 transformant lines and
non-transformant line were liquid-cultured to induce the rooting,
and then 16% sucrose was added to the culture medium. 6 weeks after
the dark-treatment, amino acid content of the microtubers was
determined.
[0176] 3 parts by weight of 80% ethanol heated to 80.degree. C. was
added to 1 part by weight of the microtubers. The obtained mixture
was ground in a mortar and then heated again to 80.degree. C. for
20 minutes. After the centrifugation at 7,000 rpm and the obtained
supernatant was recovered. After the addition of 80% ethanol, the
obtained mixture was heated to 80.degree. C. The ethanol extraction
was conducted three times, and the obtained extracts were combined
together and then 80% ethanol was added thereto to adjust the total
amount to 5 ml. After thoroughly mixing, 200 .mu.l of the extract
was taken in an Eppendrof tube, dried and then dissolved in 200
.mu.l of sterilized water. 200 .mu.l of ethyl ether was added to
the obtained solution, and they were mixed together and then
centrifuged at 12,000 rpm. The ether layer was removed. The aqueous
layer was dried again and dissolved in 400 .mu.l of 0.02 N HCl, and
the solution was filtered through a 0.45 .mu.m filter, and the
filtrate was taken as a sample and analyzed with Hitachi high-speed
amino acid analyzer (L-8800).
[0177] Amino acid analysis of microtubers derived from the
transgenic lines was conducted. At least two microtubers were
analyzed for each line, and the analytical results were
statistically treated. Glutamic acid contents of Mt2-2, Mt5-1,
Mt5-2, Mt8-1 and Mt8-2 lines from No 2, 5 and 8 plant lines into
which Mt-dAN-gdh gene had been introduced were increased to 1.7
times, 2.2 times, 2.5 times, 3.0 times and 2.2 times as high as
that of the untransformed sample, respectively (Table 11, FIG. 21).
In the plant lines into which Ct-AN-gdh gene had been introduced,
no significant difference in glutamic acid content from the
untransformed sample was recognized. As for amino acids other than
glutamic acid, a remarkable increase in glutamine content and
proline content was observed. Consequently, the total free amino
acid content was also increased to 2 to 3 times as high as that of
the untransformed plant. TABLE-US-00016 TABLE 11 Amino acid
contents in microtubers of potatoes into which Ct-AN-gdh or
Mt-dAN-gdh gene was introduced Asp Thr Ser Asn Control 1.08 .+-.
0.23 0.48 .+-. 0.13 0.66 .+-. 0.25 13.77 .+-. 6.24 CtAN-gdh no. 1-1
1.47 .+-. 0.36 0.79 .+-. 0.22 1.57 .+-. 0.21 42.12 .+-. 17.05
MtdAN-gdh no. 2-2 1.85 .+-. 0.01 0.88 .+-. 0.02 1.31 .+-. 0.03
14.79 .+-. 2.40 MtdAN-gdh no. 5-1 1.62 .+-. 0.03 0.84 .+-. 0.29
1.32 .+-. 0.54 11.12 .+-. 2.55 MtdAN-gdh no. 5-2 4.17 .+-. 3.01
0.74 .+-. 0.09 1.44 .+-. 0.02 28.62 .+-. 3.28 MtdAN-gdh no. 8-1
2.32 .+-. 0.61 0.88 .+-. 0.19 1.57 .+-. 0.24 30.97 .+-. 2.16
MtdAN-gdh no. 8-2 2.00 .+-. 0.28 0.83 .+-. 0.15 1.12 .+-. 0.13
16.69 .+-. 2.80 Glu Gln Ala His Control 2.25 .+-. 0.58 10.00 .+-.
4.25 0.52 .+-. 0.30 0.25 .+-. 0.08 CtAN-gdh no. 1-1 3.16 .+-. 1.40
17.74 .+-. 5.29 1.42 .+-. 0.59 0.80 .+-. 0.43 MtdAN-gdh no. 2-2
3.75 .+-. 0.03 27.48 .+-. 1.98 1.67 .+-. 0.03 0.26 .+-. 0.04
MtdAN-gdh no. 5-1 4.98 .+-. 1.93 34.09 .+-. 8.11 1.14 .+-. 0.51
0.59 .+-. 0.30 MtdAN-gdh no. 5-2 5.54 .+-. 0.95 25.31 .+-. 3.20
1.86 .+-. 0.17 0.36 .+-. 0.12 MtdAN-gdh no. 8-1 6.71 .+-. 2.28
20.07 .+-. 4.90 1.89 .+-. 0.25 0.46 .+-. 0.28 MtdAN-gdh no. 8-2
4.86 .+-. 1.14 14.56 .+-. 2.93 1.06 .+-. 0.19 0.28 .+-. 0.13 GABA
Arg Pro Total Control 1.29 .+-. 0.53 0.46 .+-. 0.16 5.03 .+-. 4.21
38.57 .+-. 10.31 CtAN-gdh no. 1-1 2.10 .+-. 1.19 2.93 .+-. 1.61
15.32 .+-. 9.30 93.48 .+-. 20.00 MtdAN-gdh no. 2-2 3.22 .+-. 0.52
0.78 .+-. 0.11 10.55 .+-. 1.73 71.66 .+-. 1.84 MtdAN-gdh no. 5-1
2.96 .+-. 1.74 2.00 .+-. 1.24 8.65 .+-. 5.24 73.45 .+-. 22.78
MtdAN-gdh no. 5-2 1.10 .+-. 0.21 2.18 .+-. 1.27 11.58 .+-. 0.73
86.12 .+-. 3.35 MtdAN-gdh no. 8-1 2.03 .+-. 1.15 1.53 .+-. 0.81
25.03 .+-. 1.06 98.66 .+-. 26.85 MtdAN-gdh no. 8-2 1.61 .+-. 0.47
0.68 .+-. 0.09 7.71 .+-. 2.09 55.40 .+-. 6.08 (.mu.mol/g. F.W.) (n
.gtoreq. 2)
Example 5
Investigation on the Yield of Potato Transformants
[0178] The yield of several Mt-dAN-gdh transgenic potato lines
obtained in Example 4 and the lines obtained by similar method were
tested.
[0179] The sterile transformed plants were acclimatized, and then
transferred to pots (Type 7) containing 2.5 kg of power-soil (Table
12). They were cultivated under natural light at 25.degree. C. for
2 months (in a closed system greenhouse, from March to May, 2001)
providing only with water and without top-dressing. At the end of
the period of cultivation the above-ground parts and the tuber
parts were weighed, and the number of tubers and stems were also
measured. The results were shown in Table 13 and FIGS. 22 and 23.
TABLE-US-00017 TABLE 12 Composition of power-soil Item Contents
Water 14% Distribution of particles Diameter of particle 0.5-3 mm
pH about 6.5 Amount of added fertilizer (per 1 kg) Total nitrogen
0.40 g nitrate-nitrogen 0.05 g ammonia-nitrogen 0.35 g Total
phosphate 2.00 g Water soluble potassium 0.60 g Hardly soluble
magnesia 0.20 g
[0180] TABLE-US-00018 TABLE 13 Yield of potato transformant Fresh
weight of Total weight above- of Number Number ground tuber of of
parts (g) parts (g) tubers stems Non-transformant no. 1 5.8 53.9 9
1 Non-transformant no. 2 8.2 54.7 7 1 Transformant Mt-dAN no. 1-1
56.4 175.6 14 2 Transformant Mt-dAN no. 2-3 26.4 89.0 10 2
Transformant Mt-dAN no. 3-1 53.8 228.9 15 3 Transformant Mt-dAN no.
5-3 41.6 118.0 19 2 Transformant Mt-dAN no. 8-3 64.4 224.2 16 3
A remarkable increase in the number of tubers and in the total
weight of tuber parts was observed in Mt-dAN-gdh gene introduced
potatoes.
[0181] According to the present invention, plants containing free
amino acids in a high concentration can be obtained. Thus, crops
usable as starting materials and food materials having a high
added-value are provided. According to the present invention, the
whole free amino acid content is increased 2 to 4-fold.
Particularly crops containing a very high concentration of at least
one of glutamic acid, asparagine, aspartic acid, serine, threonine,
alanine and histidine are provided. Thus, crops to be used as
starting materials having a high added-value, which do not require
the addition of these amino acids, are provided. Further, according
to the present invention, vegetables which can be directly cooked
and which contains a high concentration of glutamic acid
accumulated therein, namely food materials having a good taste, can
be provided.
[0182] In addition, the period for breeding plants containing such
free amino acids in a high concentration is remarkably shortened
according to the present invention.
[0183] Furthermore, the yield of potato can be increased according
to the present invention. The total weight of tuber parts of potato
increases at least about 1.5-fold and the number of tubers per
plant body significantly increases.
Sequence CWU 1
1
26 1 1433 DNA Aspergillus nidulans 1 atgtctaacc ttcccgttga
gcccgagttc gagcaggcct acaaggagct tgcgtcgacc 60 ctcgagaact
ccaccctctt tgagcagcac cctgaatacc gacgggctct ccaggtcgtc 120
tccgttcccg agcgcgttat ccagttccgt gtcgtttggg agaacgacaa gggcgaggtt
180 cagatcaacc gcggttaccg tgttcagttc aactccgctc tcggtcccta
caagggtggt 240 ctccgtttcc acccctccgt caacctttct atcctgaagt
tccttggctt cgagcagatc 300 ttcaaaaatg ctctcacagg acgtgcgtaa
ccgttacttc attggatgtt tgccaagagt 360 actaattggt attagtaaac
atgggtggtg gcaagggtgg ttccgacttc gaccccaagg 420 gcaagtctga
ctctgaaatt cgtcgcttct gtaccgcttt catgactgag ctctgcaagc 480
acatcggcgc ggacactgac cttcccgctg gtgatatcgg tgttactggc cgtgaggttg
540 gtttcctttt cggccagtac cgcaggatcc gcaaccagtg ggagggtgtt
ctcactggca 600 agggtggcag ctggggtggt agcttgatcc gccctgaagc
cactggatac ggtgttgtct 660 actacgttca gcacatgatc aagcacgtta
ccggtggaaa ggagtccttc gcaggcaagc 720 gtgtcgccat ctccggctcc
ggtaacgttg cccagtacgc cgctctcaag gtcatcgagc 780 tcggtggttc
cgttgtctcc ctttccgact ccaagggctc tctcattgtc aaggatgagt 840
ccgcttcttt cacccctgaa gagatcgccc tcattgccga cctcaaggtt gcccgcaagc
900 aactctccga gctcgccacc tcctccgctt tcgccggcaa gttcacctac
atccccgatg 960 ctcgcccttg gaccaacatt cccggcaagt tcgaggttgc
tctcccttct gccactcaga 1020 acgaagtctc cggcgaggaa gccgagcacc
tcatcaagtc cggtgtccgc tatattgctg 1080 agggttccaa catgggttgc
acccaggccg ccatcgacat ctttgaggct caccgcaacg 1140 ccaaccccgg
cgatgccatc tggtacgccc ctggtaaagc cgccaacgct ggtggtgtcg 1200
ccgtctctgg tcttgagatg gctcagaact ctgctcgtct ctcctggaca tccgaggagg
1260 tcgatgctcg cctcaagggc atcatggagg actgcttcaa gaacggtctc
gagactgctc 1320 agaagttcgc tactcctgcc aagggcgtcc tgccttccct
cgtcaccggt tccaacattg 1380 ccggtttcac caaggtcgcc gaggccatga
aggaccaggg tgactggtgg tga 1433 2 1240 DNA Lycopersicon esculentum 2
atgaatgctt tagcagcaac taatagaaat tttaagctgg cagctaggct tcttggttta
60 gactcaaagt tggaactaag tctgctaatc cctttcagga aattaaggtg
gagtgtacta 120 taccgaagga tgatggcaca ttggcatctt ttgttggatt
cagggtacag cacgacaatg 180 cacgagggcc tatgaaaggc ggaatcagat
accacccgga ggttgatcct gatgaggtga 240 atgcattagc acagctaatg
acatggaaga cagcggtcgc caatattacc atatggtggg 300 gctaaaggag
gaataggatg tagtcctagt gacctgagta tctctgagtt ggaacgactt 360
actcgagtat ttactcaaaa aatacatgac ctaatcggaa ttcacaccga tgttcctgca
420 ccagatatgg gaacaaatcc tcagacaatg gcatggattt tagacgagta
ctcaaaattt 480 catggttatt cacctgctgt ggtaactgga aaacctgttg
atctcggtgg atctctaggc 540 agagatgcag ctactggaag ggggggctct
ctttgctaca gaagccctgc ttaatgagca 600 tgggaagagt gttgctggtt
cagcgttttg ttatacaggg atttggtaat gttggttcct 660 gggctgcaaa
actcatccat gagcaaggtg ggaaagttgt agcagtgagt gacataactg 720
gtgccataaa gaatgagaag ggaatcgaca tagaaagcct attcaaacac gtgaaggaaa
780 ctcgtggagt taaaggtttc catgatgcac atccaattga tgcaaattca
atactggtag 840 aagactgtga tgttcttatc ccagctgccc tcggtggagt
aatcaacaag gataaccaca 900 aattgaaaat taaagccaaa tatattattg
aggctgctaa ccatccaact gatccagaag 960 ctgatgagat ttgtcaaaga
aaggagtcac catcctaccg gatatttatg ccaactcggg 1020 tggtgtcacc
gtcagttatt ttgagtgggt ccagaacatc caaggcttta tgtgggatga 1080
gaaaaaagtg aatgatgagt tgaagacata catgacaaga ggttttaaag atgtcaagga
1140 tatgtgcaag actcacaact gtgacctccg aatgggcgcc ttcaccttag
gtgttaaccg 1200 tgtagctaga gcaaccgttc ttcgaggatg ggaggcgtaa 1240 3
28 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 3 tctagaatgt ctaaccttcc
cgttgagc 28 4 28 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 4 gagctctcac
caccagtcac cctggtcc 28 5 27 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 5
tctagaatga atgctttagc agcaact 27 6 27 DNA ARTIFICIAL SEQUENCE
SYNTHETIC DNA 6 gagctcttac gcctcccatc ctcgaag 27 7 28 DNA
ARTIFICIAL SEQUENCE SYNTHETIC DNA 7 tctagaatgt ctaaccttcc cgttgagc
28 8 24 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 8 cacccatgtt
tagtcctgtg agag 24 9 24 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 9
ctctcacagg actaaacatg ggtg 24 10 28 DNA ARTIFICIAL SEQUENCE
SYNTHETIC DNA 10 gagctctcac caccagtcac cctggtcc 28 11 26 DNA
ARTIFICIAL SEQUENCE SYNTHETIC DNA 11 ggatccatga atgctttagc agcaac
26 12 26 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 12 tctagataaa
ccaagaagcc tagctg 26 13 30 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 13
ctgcagatgg cttcctcaat tgtctcatcg 30 14 30 DNA ARTIFICIAL SEQUENCE
SYNTHETIC DNA 14 tctagagcat ctaacgcgtc caccattgct 30 15 26 DNA
ARTIFICIAL SEQUENCE SYNTHETIC DNA 15 tctagaatga atgctttagc agcaac
26 16 30 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 16 gggaaggtta
gacattaaac caagaagcct 30 17 30 DNA ARTIFICIAL SEQUENCE SYNTHETIC
DNA 17 aggcttcttg gtttaatgtc taaccttccc 30 18 26 DNA ARTIFICIAL
SEQUENCE SYNTHETIC DNA 18 gagctcttac gcctcccatc ctcgaa 26 19 30 DNA
ARTIFICIAL SEQUENCE SYNTHETIC DNA 19 ctgcagatgg cttcctcaat
tgtctcatcg 30 20 24 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 20
aaggttagac atgcatctac cgcg 24 21 24 DNA ARTIFICIAL SEQUENCE
SYNTHETIC DNA 21 cgcgttagat gcatgtctaa cctt 24 22 26 DNA ARTIFICIAL
SEQUENCE SYNTHETIC DNA 22 gagctcttac gcctcccatc ctcgaa 26 23 26 DNA
ARTIFICIAL SEQUENCE SYNTHETIC DNA 23 aagcttatat aacccaaaat atacta
26 24 26 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 24 tctagaggta
ccattaattg ctaatt 26 25 21 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 25
cccctcggta tccaattaga g 21 26 24 DNA ARTIFICIAL SEQUENCE SYNTHETIC
DNA 26 cggggggtgg gcgaagaact ccag 24
* * * * *