U.S. patent application number 12/000080 was filed with the patent office on 2009-03-12 for soybean mutant strain and soybean oil therefrom.
Invention is credited to Toyoaki Anai, Yutaka Takagi.
Application Number | 20090068658 12/000080 |
Document ID | / |
Family ID | 30428279 |
Filed Date | 2009-03-12 |
United States Patent
Application |
20090068658 |
Kind Code |
A1 |
Anai; Toyoaki ; et
al. |
March 12, 2009 |
Soybean mutant strain and soybean oil therefrom
Abstract
A soybean oil of a decreased content of polyunsaturated fatty
acids is produced by hybridizing, selecting and raising a soybean
mutant KK21 characterized by a gene having a sequence of base given
by the sequence No. 1 of the sequence listing, a soybean mutant M23
characterized by deletion of a gene having a sequence of base given
by the sequence No. 2 of the sequence listing, a soybean mutant M24
characterized by a gene having a sequence of base given by the
sequence No. 3 of the sequence listing, a soybean mutant M5
characterized by a gene having a sequence of base given by the
sequence No. 4 of the sequence listing, a soybean mutant J18
characterized by deletion of a gene having a sequence of base given
by the sequence No. 5 of the sequence listing, and one or two kinds
or more of these soybean mutants.
Inventors: |
Anai; Toyoaki; (Saga-shi,
JP) ; Takagi; Yutaka; (Saga-shi, JP) |
Correspondence
Address: |
WENDEROTH, LIND & PONACK, L.L.P.
2033 K STREET N. W., SUITE 800
WASHINGTON
DC
20006-1021
US
|
Family ID: |
30428279 |
Appl. No.: |
12/000080 |
Filed: |
December 7, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10419865 |
Apr 22, 2003 |
7326547 |
|
|
12000080 |
|
|
|
|
Current U.S.
Class: |
435/6.11 ;
435/6.12; 554/1; 800/312 |
Current CPC
Class: |
C12N 9/0083 20130101;
C12N 15/8247 20130101 |
Class at
Publication: |
435/6 ; 800/312;
554/1 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; A01H 5/00 20060101 A01H005/00; C07C 57/00 20060101
C07C057/00 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 22, 2002 |
JP |
2002-119821 |
Claims
1. A soybean mutant strain capable of fruiting beans of a decreased
content of polyunsaturated fatty acids, wherein the mutant
comprises a genome lacking the nucleotide sequence of SEQ ID NO: 2
or SEQ ID NO: 5.
2. A soybean oil of a decreased content of polyunsaturated fatty
acids as an oleaginous ingredient in the beans of the soybean
mutant strain defined in claim 1.
3. A method for assay of a mutant gene derived from the mutant
strain KK21 which comprises the steps of: amplifying a part of the
base sequence of the gene GmFAD2-1 by the PCR reaction using the
primer 1: 5'-attgatagcccctccgttcccaaga-3' (SEQ ID NO: 6) and the
primer 2: 5'-attgtgagtgtgacgagaagagaaac-3' (SEQ ID NO: 7); and
analyzing for the target base sequence by using the primer 3:
5'-gggtctagcaaaggaaacaacaatgggaggt-3' (SEQ ID NO: 8).
4. (canceled)
5. A method for assay of a mutant gene derived from the mutant
strain J18 which comprises the steps of: amplifying simultaneously
a part of the base sequence of the gene GmFAD3-1a and a part of the
base sequence of the gene GmFAD3-1b by the PCR reaction using the
primer 5: 5'-ttattacgcaccacccaccacgtatccct-3' (SEQ ID NO: 10) and
the primer 6: 5'-gttgcgagtggaggagcagagaatcagtc-3' (SEQ ID NO: 11);
and analyzing for the target base sequence by a combination of
digestion with the restriction enzyme Kpn I and the method of
electrophoresis.
6. A method for assay of the mutant gene derived from the mutant
strain M5 or the mutant strain derived from the mutant strain M24
which comprises the steps of: amplifying simultaneously a part of
the base sequence of the gene GmFAD3-1a and a part of the base
sequence of the gene GmFAD3-1b by the PCR reaction using the primer
5: 5'-ttattacgcaccacccaccacgtatccct-3' (SEQ ID NO: 10) and the
primer 6: 5'-gttgcgagtggaggagcagagaatcagtc-3' (SEQ ID NO: 11);
conducting separation of the target base sequences by a combination
of digestion with the restriction enzyme of Kpn I and the method of
electrophoresis; and analyzing for the base sequence by using the
primer 7: 5'-gtggatcgtgactatggttggatcta-3' (SEQ ID NO: 12).
7. A soybean mutant strain capable of fruiting beans of a decreased
content of polyunsaturated fatty acids, wherein the mutant
comprises a genome having a nucleotide sequence of SEQ ID NO: 1,
SEQ ID NO: 3 or SEQ ID NO: 4.
Description
[0001] This application is a divisional of application Ser. No.
10/419,865, filed Apr. 22, 2003, now allowed. The teachings of the
above-referenced application is hereby incorporated by
reference.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to a novel mutant strain
obtained by irradiation with X-rays using a soybean strain Bay as a
new strain, and a soybean oil of a decreased content of
polyunsaturated fatty acids obtained therefrom.
[0003] In general, a triacylglycerol which is a main component of
fats and oils present in plants, particularly their seeds, is a
compound obtained by bonding three fatty acid molecules to one
glycerol molecule, and depending on the kind of the bonded fatty
acid, physical and chemical properties and nutritive values are
different. The composition of the fatty acids is specific to plant
species from which they are derived, and therefore, it is
impossible to avoid limitation of its use for each plant
species.
[0004] On the other hand, for suppressing degeneration by oxidation
in storage of this plant fat and oil, it is necessary to decrease
the content of polyunsaturated fatty acids liable to be
oxidized.
[0005] It is known that, in a soybean seed, a biosynthesis system
of palmitic acid.fwdarw.stearic acid.fwdarw.oleic
acid.fwdarw.linolic acid.fwdarw.linolenic acid is present, and
conversion from oleic acid into linolic acid is conducted with a
microsomal .omega.-6 fatty acid desaturase enzyme and conversion
from linolic acid into linolenic acid is conducted with a
microsomal .omega.-3 fatty acid desaturase enzyme, and that
.omega.-6 and .omega.-3 fatty acid desaturations with these enzymes
are correlated with a plurality of genes, and known that parts of
them are controlled by genes at the FAD 2-position and FAD
3-position [Plant Physiol., (1993), vol. 103, pages 467-476 and
Theor. Appl. Genet., (1997), vol. 94, pages 356-359].
SUMMARY OF THE INVENTION
[0006] The present invention has been accomplished intending a
mutant usable in strain-improvement to decrease the content of
polyunsaturated fatty acids in a soybean seed and production of a
soybean oil of a decreased content of polyunsaturated fatty acids
using the above-mentioned mutant as a raw material.
[0007] The present inventors have intensively studied for obtaining
a soybean strain which gives a soybean oil of improved quality, and
resultantly found that a mutant obtained by irradiation with X-rays
using a soybean strain Bay as a new strain allows production of an
oil of a decreased content of linolic acid and linolenic acid,
leading to completion of the invention based on this finding.
[0008] Namely, the present invention provides a method of producing
a soybean oil of a decreased content of polyunsaturated fatty acids
comprising: hybridizing, selecting and raising a soybean mutant
KK21 characterized by a gene having a sequence of base given by the
sequence No. 1 of the sequence listing, a soybean mutant M23
characterized by deletion of a gene having a sequence of base given
by the sequence No. 2 of the sequence listing, a soybean mutant M24
characterized by a gene having a sequence of base given by the
sequence No. 3 of the sequence listing, a soybean mutant M5
characterized by a gene having a sequence of base given by the
sequence No. 4 of the sequence listing, a soybean mutant J18
characterized by deletion of a gene having a sequence of base given
by the sequence No. 5 of the sequence listing, and one or two kinds
or more of these soybean mutants.
BRIEF DESCRIPTION OF THE DRAWING
[0009] FIG. 1 is a view showing the analysis pattern of a gene of a
soybean mutant KK21.
[0010] FIG. 2 is a view showing the analysis pattern of a gene of a
soybean mutant M23 and other genes compared with it according to a
Southern blotting method.
[0011] FIG. 3 is a view showing the analysis pattern of a gene of a
soybean mutant M23 and other genes compared with it according to a
RT-PCR method.
[0012] FIG. 4 is a view showing the analysis pattern of a gene of a
soybean mutant M24.
[0013] FIG. 5 is a view showing the analysis pattern of a gene of a
soybean mutant M5.
[0014] FIG. 6 is a view showing the analysis pattern of a gene of a
soybean mutant J18 and other genes compared with it according to a
Southern blotting method.
[0015] FIG. 7 is a view showing the analysis pattern of a gene of a
soybean mutant J18 and other genes compared with it according to a
RT-PCR method.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0016] The mutant gene of the present invention having a sequence
of base given by the sequence No. 1 of the sequence listing
characterizing a soybean mutant KK21 is obtained by deletion of
thymine (t), the 233rd base from the initiation codon, in FAD2-1
which is one of microsomal .omega.-6 fatty acid desaturase enzyme
genes in soybean, and because of this reason, one of enzymes
necessary for synthesis of linolic acid is not formed normally. As
the feature of a soybean mutant M23, expression of a FAD2-1 gene
having a sequence of base given by the sequence No. 2 of the
sequence listing is not found, and one of necessary enzymes is not
synthesized. The mutant gene having a sequence of base given by the
sequence No. 3 of the sequence listing characterizing a soybean
mutant M24 is obtained by deletion of adenine (a), 1078th base from
the initiation codon, in FAD3-1a which is one of microsomal
.omega.-3 fatty acid desaturase enzyme genes in soybean, and
because of this reason, one of enzymes necessary for synthesis of
linolenic acid is not formed normally. The mutant gene having a
sequence of base given by the sequence No. 4 of the sequence
listing characterizing a soybean mutant M5 is obtained by deletion
of bases from the 1033rd base, cytosine (c), to the 1051st base,
guanine (g) from the initiation codon in FAD3-1b, and because of
this reason, one of enzymes necessary for synthesis of linolenic
acid is not formed normally. Further, as the feature of a soybean
mutant J18, expression of a FAD3-1b gene having a sequence of base
given by the sequence No. 5 of the sequence listing is not found,
and one of necessary enzymes is not synthesized.
[0017] These soybean mutants are obtained by irradiating 4000
parent soybean strain Bays with X-rays at an intensity of 1
Sv/minute for 200 minutes at a total dosage of about 200 Sv
(sievert) to induce mutation.
[0018] Then, seeds thus irradiated with X-rays, namely, irradiated
first generation seeds (hereinafter, referred to as M1 seed) are
disseminated on the field and cultivated to obtain self-propagating
progeny, namely, irradiated second generation seeds (hereinafter,
referred to as M2 seed). Then, irradiated third generation seeds
(hereinafter, referred to as M3 seed) are raised from the
irradiated M2 seeds in the same manner, and the composition of
fatty acid is analyzed for each individual of M2 seeds. Thus, from
the analysis results of the fatty acid compositions, it was
confirmed that on average 4 to 5 mutants are generated from about
2000 M2 seeds, and mutants were separated. Regarding irradiated M4
generation and followings obtained from the thus obtained M3 seeds,
experiments confirming mutants were conducted sequentially to
obtain respective mutants.
[0019] In the present invention, detection of these respective
genes can be conducted by a RT-PCR (reverse transcribed-polymerase
chain reaction) method, a combination of this method with a dideoxy
method, or a Southern blotting method, as shown in the following
methods (1) to (5).
(1) Detection of Variant FAD2-1 Gene Generated in KK21 Line (High
Oleic Acid Mutant Line):
[0020] This can be conducted, by combining a RT-PCR method with a
dideoxy method, as follows. Namely, all RNAs are extracted from a
plant material (immature seed) according to an ordinary method,
then, a cDNA is produced using an oligo-dT primer according to an
ordinary method. Next, using this cDNA as a template, a PCR
reaction is conducted according to an ordinary method using a
primer 1: 5'-attgatagcccctccgttcccaaga-3' (SEQ ID NO: 6) and a
primer 2: 5'-attgtgagtgtgacgagaagagaaac-3' (SEQ ID NO: 7) to
amplify a part of a sequence of base of a FAD2-1 gene, then, using
the purified DNA fragment as a template, the analysis of a sequence
of base is conducted according to an ordinary method using a primer
3: 5'-gggtctagcaaaggaaacaacaatgggaggt-3' (SEQ ID NO: 8). As a
result, identification of a variant FAD2-1 gene derived from the
KK21 line is possible if deletion of a base t shown in FIG. 1 is
observed.
(2) Detection of Variant FAD2-1 Gene Generated in M23 Line (High
Oleic Acid Mutant Line):
[0021] (a) Detection by Southern blotting method; DNA obtained by
extraction according to an ordinary method from a plant material
(any tissue of leaf, seed, root and the like is possible) is
digested using a restriction enzyme EcoR I, and analyzed by a
Southern blotting method according to an ordinary method. In this
operation, a FAD2-1 gene is used as a probe, however, labeling of a
probe may be conduced by any of methods using a radioactive isotope
or methods using chemical modifications, without causing problems.
In a Bay (parent strain) having a normal FAD2-1 gene, three bands
of about 1.9 kbp, 2.5 kbp and 4.6 kbp are detected, and when a
variant FAD2-1 gene derived from M23 line is present, this can be
identified by disappearance of a band of about 4.6 kbp. Analysis
pattern by this method is shown in FIG. 2.
[0022] (b) Detection by RT-PCR method; All RNAs are extracted from
a plant material (immature seed) according to an ordinary method,
then, a cDNA is produced using an oligo-dT primer according to an
ordinary method. Next, using this cDNA as a template, a PCR
reaction is conducted according to an ordinary method using a
primer 1: 5'-attgatagcccctccgttcccaaga-3' (SEQ ID NO: 6) and a
primer 4: 5'-atacacacaaagtcattacgcggcaa-3' (SEQ ID NO: 9) to
amplify a sequence of base including the overall length of a
protein coding region of a FAD2-1 gene. When this reaction product
is analyzed by an agarose electrophoresis method according to an
ordinary method, one band of about 1.3 kbp is detected in a Bay
(parent strain) having a normal FAD2-1 gene, while in the case of a
variant FAD2-1 gene derived from M23 line, this can be identified
by utterly no detection of bands. The analysis data by this method
are shown in FIG. 3.
(3) Detection of Variant FAD3-1a Gene Generated in M24 Line (Low
Linolenic Acid Mutant Line):
[0023] This can be conducted, by combining a RT-PCR method with a
dideoxy method, as follows. All RNAs are extracted from a plant
material (immature seed) according to an ordinary method, then, a
cDNA is produced using an oligo-dT primer according to an ordinary
method. Next, using this cDNA as a template, a PCR reaction is
conducted according to an ordinary method using a primer 5:
5'-ttattacgcaccacccaccacgtatccct-3' (SEQ ID NO: 10) and a primer 6:
5'-gttgcgagtggaggagcagagaatcagtc-3' (SEQ ID NO: 11) to amplify
sequences of base containing the overall lengths of protein coding
regions of FAD3-1a and FAD3-1b genes simultaneously. Then, DNA
fragments purified according to an ordinary method are digested
using a restriction enzyme Kpn I, and only a FAD3-1a is separated
from a DNA fragment of about 1.2 kbp and purified using agarose
gel, then, using this as a template, the analysis of a sequence of
base is conducted according to an ordinary method using a primer 7:
5'-gtggatcgtgactatggttggatcta-3' (SEQ ID NO: 12). The analysis
pattern in this operation is shown in FIG. 4. As a result,
identification of a variant FAD3-1a gene derived from the M24 line
is possible if deletion of a base a shown in FIG. 4 is
observed.
(4) Detection of Variant FAD3-1b Gene Generated in M5 Line (Low
Linolenic Acid Mutant Line):
[0024] This can be conducted, by combining a RT-PCR method with a
dideoxy method, as follows. All RNAs are extracted from a plant
material (immature seed) according to an ordinary method, then, a
cDNA is produced using an oligo-dT primer according to an ordinary
method. Next, using this cDNA as a template, a PCR reaction is
conducted according to an ordinary method using a primer 5:
5'-ttattacgcaccacccaccacgtatccct-3' (SEQ ID NO: 10) and a primer 6:
5'-gttgcgagtggaggagcagagaatcagtc-3' (SEQ ID NO: 11) to amplify
sequences of base containing the overall lengths of protein coding
regions of FAD3-1a and FAD3-1b genes simultaneously. Then, DNA
fragments purified according to an ordinary method are digested
using a restriction enzyme Kpn I, and a DNA fragment of about 0.3
kbp only of a FAD3-1b gene is separated and purified using agarose
gel, then, using this as a template, the analysis of a sequence of
base is conducted according to an ordinary method using a primer 7:
5'-gtggatcgtgactatggttggatcta-3' (SEQ ID NO: 12). The analysis
pattern in this operation is shown in FIG. 5. As a result,
identification of a variant FAD3-1b gene derived from the M5 line
is possible if deletion of 19 bases (5'-cttcgtaagtgacactgga-3')
(SEQ ID NO: 13) shown in FIG. 5 is observed.
(5) Detection of Variant FAD3-1b Gene Generated in J18 Line (Low
Linolenic Acid Mutant Line):
[0025] (a) Detection by Southern blotting method; DNA obtained by
extraction according to an ordinary method is digested using a
restriction enzyme EcoR I, and analyzed by a Southern blotting
method according to an ordinary method. In this operation, a
FAD3-1b gene is used as a probe, however, labeling of a probe may
be conduced by any of methods using a radioactive isotope or
methods using chemical modifications, without causing problems. In
a Bay (parent strain) having a normal FAD3-1b gene, four bands of
about 0.35 kbp, 2.1 kbp, 2.3 kbp and 4.3 kbp are detected, and when
a variant FAD3-1b gene derived from J18 line is present, this can
be identified by disappearance of two bands of about 0.35 kbp and
4.3 kbp. Analysis pattern by this method is shown in FIG. 6.
[0026] (b) Detection by RT-PCR method; All RNAs are extracted from
a plant material (immature seed) according to an ordinary method,
then, a cDNA is produced using an oligo-dT primer according to an
ordinary method. Next, using this cDNA as a template, a PCR
reaction is conducted according to an ordinary method using a
primer 5: 5'-ttattacgcaccacccaccacgtatccct-3' (SEQ ID NO: 10) and a
primer 6: 5'-gttgcgagtggaggagcagagaatcagtc-3' (SEQ ID NO: 11) to
amplify sequences of base containing the overall lengths of protein
coding regions of FAD3-1a and FAD3-1b genes simultaneously. DNA
fragments purified according to an ordinary method are digested
using a restriction enzyme Kpn I, and DNA fragments are separated
using agarose gel. As a result, in Bays (parent strains) having
normal FAD3-1a and FAD3-1b genes, a DNA fragment of about 1.2 kbp
derived from a FAD3-1b gene and DNA fragments of about 900 bp and
300 bp derived from FAD3-1a gene are detected, while in the case of
a variant FAD3-1b gene derived from a J18 line, this can be
identified by disappearance of a DNA fragment of about 1.2 kbp of
them. Analysis data of this method are shown in FIG. 7.
[0027] Next, the present invention will be illustrated further in
detail by examples, but the scope of the invention is not limited
by these examples at all.
[0028] In the following examples, a parent Bay and, three mutants
M24, M5 and J18 of low linolenic acid content obtained by
irradiation on the Bay with X-rays, were cultivated in greenhouses
of Saga University, Department of Agriculture, and leaves were
sampled, then, frozen in liquid nitrogen and stored in a freezer of
-80.degree. C., before using as a plant material.
EXAMPLES
(1) Extraction of Genomic DNA
[0029] Frozen leaves were ground in a mortar, then, to 1 g of this
powder was added 3 ml of 2% CTAB buffer [10 mM Tris-HCl (pH 8.0),
1.4 M NaCl, 20 mM EDTA, 2% CTAB], and the mixture was stirred
gently. This was incubated on a water bath of 65.degree. C. for 30
minutes and the same volume of chloroform was added and stirred
gently, then, centrifugal separation was conducted at 4.degree. C.
under 8000 rpm for 10 minutes to remove proteins, and again,
proteins in the aqueous layer were removed by extraction with
chloroform. This aqueous layer was transferred to another
centrifugal tube, and 1% CTAB precipitation buffer [50 mM Tris-HCl
(pH 8.0), 1% CTAB] was added so that the total amount was 12 ml and
they were mixed gently, then, centrifugal separation was conducted
at 4.degree. C. under 8000 rpm for 10 minutes to precipitate a
CTASB-DNA complex. The supernatant was discarded, then, the
precipitate was dissolved in 4 ml of 1M NaCl for removal of CTAB,
and 4 ml of isopropanol was added, and centrifugal separation was
conducted at 4.degree. C. under 8000 rpm for 10 minutes to recover
DNA. This precipitate was washed with 70% ethanol, then, dissolved
in 200 .mu.l of TE buffer [10 mM Tris-HCl (pH 8.0), 1 mM EDTA]
containing RNase A (10 .mu.g/.mu.l), and incubated at 37.degree. C.
for 1 hour to digest RNA. For removal of proteins, to this was
added the same volume of TLE saturated phenol-chloroform (1:1), and
centrifugal separation was conducted at 4.degree. C. under 15000
rpm for 5 minutes, the aqueous layer was moved to a new centrifugal
tube of 1.5 ml and an extraction operation was conducted again,
then, phenol in the aqueous layer was removed using only
chloroform. These samples were subjected to electrophoresis
together with EDNA not cut, the concentration was measured, then,
the mixture was controlled so that the DNA concentration was 0.2
.mu.g/.mu.l and stored at -20.degree. C.
(2) Preparation of Total RNA
[0030] According to a method obtained by improving a phenol/SDS
method, soybean seeds sampled at four stages (5 mm or less, 5 to 7
mm, 7 to 10 mm and 10 mm or more) were frozen in liquid nitrogen,
and ground in a mortar, then, to 5 g of this powder was added 25 ml
of extraction buffer [180 mM Tris-HCl (pH 8.2), 90 mM LiCl, 4.5 mM
EDTA, 1% SDS] and 10 ml of water-saturated phenol, and the mixture
was stirred using a polytron type homogenizer at a maximum speed
for about 1 minute. Further, 9 ml of chloroform was added and
stirred sufficiently, and subjected to centrifugal separation at
4.degree. C. under 8000 rpm for 10 minutes. This aqueous layer was
moved to a new centrifugal tube, and to this was added 5 ml of 2 M
NaOAc (pH 4.0) and each 5 ml of water-saturated phenol and
chloroform and the mixture was stirred sufficiently, and subjected
to centrifugal separation at 4.degree. C. under 8000 rpm for 10
minutes. Thereafter, extraction with 10 ml of water-saturated
phenol and chloroform was repeated six times in total, then, the
supernatant was extracted with 10 ml of chloroform to remove
phenol. 15 ml of 8 M LiCl was added to this aqueous layer, and the
mixture was stirred gently up and down, then, allowed to stand
still overnight on ice to precipitate a lithium salt of RNA, and
subjected to centrifugal separation at 4.degree. C. under 8000 rpm
for 10 minutes to recover the precipitate. The thus obtained
precipitate was dissolved in 500 .mu.l of sterile water, moved to a
1.5 ml centrifugal tube, then, 3 M NaOAc of 1/10 amount was added
and cooled on ice for 30 minutes, then, subjected to centrifugal
separation at 4.degree. C. under 15000 rpm for 5 minutes, to remove
polysaccharides contaminated. 750 .mu.l of isopropanol
corresponding to 1.5-fold amount was poured into a 1.5 ml
centrifugal tube and the supernatant was added into this tube, and
subjected to centrifugal separation at 4.degree. C. under 15000 rpm
for 5 minutes, then, the supernatant was discarded and the
precipitate was dissolved in 500 .mu.l of sterile water.
(3) Purification of cDNA
[0031] A reaction solution of a total amount of 20 .mu.l containing
50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl.sub.2, 10 mM DDT, 300
.mu.M dNTPs, 0.5 .mu.g of Oligo-dT primer and 200 units of M-MLV
reverse transcriptase (manufactured by GIBCO-BRL) was incubated for
1 hour at 37.degree. C. to synthesize cDNA from 5 .mu.g of total
RNA. Next, using 1 .mu.l of this 10-fold-diluted cDNA solution as a
template, 20 .mu.l of a PCR reaction solution was prepared
containing 0.4 .mu.l of Advantage II polymerase (manufactured by
CRONTECH), 1.times. Advantage II buffer, 1.times.GC-RICH solution,
250 .mu.M dNTPs and 0.1 pmol of a primer, and a PCR reaction was
conducted. As conditions for the PCR reaction, one cycle of
incubation at 95.degree. C. for 2 minutes was conducted,
subsequently, reaction cycle incubation at 95.degree. C. for 30
seconds, at 65.degree. C. for 30 seconds and at 72.degree. C. for 3
minutes was repeated 35 times. The thus obtained PCR product was
subjected to electrophoresis treatment at 100 V for 30 minutes
using 1% agarose gel, and the intended cDNA fragment (Gm FAD3-1:
about 1.2 kbp, Gm FAD3-2: about 1.3 kbp) detected by staining with
ethidium bromide was recovered from the gel using QIAEX II Gel
Extraction Kit (manufactured by QIAGEN).
(4) Production of RNA Probe
[0032] To 1 .mu.l of a plasmid (pGEM-T Easy Vecter), 5 .mu.l of T4
DNA Ligase, 1.times. Rapid Ligation buffer and 1 .mu.l of T4 DNA
Ligase was added 3 .mu.l of purified FAD3-1 or FAD3-2 gene
fragment, and the mixture was incubated at 12.degree. C. overnight.
The ligated plasmid was transformed into E. coli (XL-10 Gold),
then, plated on a LB agar medium containing 50 mg/l of
carbenicillin, 40 mg/l of X-gal and 200 .mu.M of IPTG, and cultured
overnight at 37.degree. C., then, white colonies were selected, and
incorporation and direction of fragments were confirmed using a PCR
method. Bacteria holding the intended plasmid were inoculated on a
LB liquid medium containing carbenicillin and cultured at
37.degree. C. for about 14 hours, then, the plasmid was extracted.
A PCR reaction was conducted using the thus obtained plasmid as a
template to amplify DNA fragments containing a multi-cloning site,
and purified by agarose electrophoresis. This DNA fragment was
subjected to a protease treatment [0.5% SDS, 20 .mu.m/ml protease]
at 37.degree. C. for 2 hours to completely digest RNase mixed in
the DNA solution, and the protease was removed by extraction twice
with phenol-chloroform. To this supernatant was added 75 .mu.l of
isopropanol and 5 .mu.l of 3 M NaOAc and the mixture was cooled at
-80.degree. C. for 15 minutes, then, subjected to centrifugal
separation at 4.degree. C. under 15000 rpm for 10 minutes, and the
thus obtained precipitate was dissolved in 20 .mu.l of sterile
water. A RNA probe labeled with DIG RNA Labeling Mix (manufactured
by Roche) was produced using this DNA as a template.
(5) Southern Blotting Analysis
[0033] Genomic DNA was completely digested by EcoR I, and separated
depending on the molecular weight by 1% agarose gel
electrophoresis. A DNA fragment on this gel was transferred with an
alkali solution [1.5 M NaCl, 0.5 M NaOH], and transferred to
Biodyne plus membrane (manufactured by PALL). Thereafter,
pre-hybridization was conducted at 50.degree. C. for 5 hours or
more in high SDS concentration hybridization buffer [50% formamide,
5.times.SSC, 0.1% N-lauroylsarcosine, 0.02% SDS, 1% blocking
reagent], and RNA probes for DIG-labeled Gm FAD3-1 and Gm FAD3-2
were added, further, hybridization was conducted overnight at
50.degree. C. Then, the membrane was washed twice in an aqueous
solution containing 2.times.SSC and 0.1% SDS each for 5 minutes,
further, washed three times in an aqueous solution containing
0.1.times.SSC and 0.1% SDS at 65.degree. C. each for 20 minutes, to
remove excess probes adhered to the membrane, then, washed with
washing buffer [0.1 M maleic acid, 0.15 M NaCl, 0.3% Tween 20 (pH
7.5)] of room temperature for removal of SDS for 5 minutes. Then, a
blocking reaction was conducted for 1 hour in buffer 2 [0.1 M
maleic acid, 0.15 M NaCl, 0% blocking reagent (pH 7.5)], and
incubation was conducted for 1 hour in the buffer 2 containing
1000-fold-diluted Anti-Digoxigenin-APFAB fragments (manufactured by
Roche), to bond a label antibody to the DIG-labeled probe.
Thereafter, washing in washing buffer was conducted three times
each for 15 minutes, to remove excess antibodies. This was
equilibrated in buffer 3 [10 mM Tris-HCl, 10 mM NaCl, 1 mM
MgCl.sub.2], then, incubation was conducted for 5 minutes in a
CDP-Star solution (manufactured by Roche), and the membrane was
sealed with a wrapping and incubated at 37.degree. C. for 15
minutes. Chemoluminescence was detected by allowing it to be
sensitized on an X-ray film.
(6) Northern Blotting Analysis
[0034] 5 .mu.g of total RNA was separated by electrophoresis using
1.2% formaldehyde.cndot.agarose gel [1.times.MOPS buffer, 5%
formaldehyde, 1.2% agarose], and transferred onto Biodyne plus
membrane (manufactured by PALL) by using 20.times.SSC buffer. The
membrane was rinsed with 2.times.SSC, then, irradiated with UV
light of 70 mJ/cm.sup.2 using UV cross-linker (manufactured by
Amersham Pharmacia) to fix RNA. Hybridization was conducted at
65.degree. C. using high SDS concentration hybridization buffer, in
the same manner as for Southern blotting analysis, and detection
was conducted also in the same manner.
(7) Sequence Analysis
[0035] Analysis of a sequence of base was conducted using Big Dye
Terminator v3.0 Ready Reaction Cycle sequencing Kit (manufactured
by Applied Biosystems). Using QIAEX II Gel Extraction Kit
(manufactured by QIAGEN) as a template, a sequence reaction
solution was produced containing cDNA fragments recovered from gel
(cDNA fragment: template concentration, 100 to 200 bp: 2 ng/.mu.l,
200 to 500 bp: 5 ng/.mu.l, 500 to 1000 bp: 10 ng/.mu.l, 1000 to
2000 bp: 20 ng/.mu.l, >2000 bp: 50 ng/.mu.l), 1.times.
Terminator Ready Reaction PreMix, 1.times. sequencing buffer [80 mM
Tris-HCl (pH 9.0), 2 mM MgCl.sub.2, 0.01% BSA] and 1 .mu.mol/.mu.l
primer, and incubation at 95.degree. C. for 30 seconds was
conducted once, then, a reaction cycle at 95.degree. C. for 30
seconds and at 60.degree. C. for 4 minutes was repeated 30 times,
to effect a sequence reaction. This reaction solution was subjected
to purification of the reaction product using a spin column
prepared by filling Multiscreen HV plate (manufactured by
MILLIPORE) with Sephadex G-50 fine (manufactured by Amarsham
Pharmacia), and the purified substance was dried to solid in an
oven at 65.degree. C. This was dissolved completely with 30 .mu.l
of Template suspension reagent, and heated at 95.degree. C. for 10
minutes for thermal denaturation, then, rapidly chilled taking 10
minutes with ice water, and the produced was analyzed using ABI
PRISM 310 Genetic Analyzer.
[0036] Sequences of base of genes of thus obtained mutants M24, M5
and J18 are given by the sequence Nos. 3, 4 and 5 of the sequence
listing.
[0037] Next, for producing a soybean oil of a decreased content of
polyunsaturated fatty acids according to the present invention,
soybean mutant strains obtained as described above are hybridized,
selected and raised, to cause proliferation and cultivation,
obtaining seeds. In this operation, it is optional that the mutant
contains a single kind of gene or contains two or more kinds of
genes.
[0038] In the cases of soybean mutants KK21 and M23, a microsomal
.omega.-6 fatty acid desaturase enzyme producing linolic acid from
oleic acid is deficient, therefore, production of linolic acid and,
linolenic acid obtained by further change of linolic acid is
suppressed, obtaining a soybean oil of a decreased content of
linolic acid and linolenic acid, and in the cases of soybean
mutants M24, M5 and J18, a microsomal .omega.-3 fatty acid
desaturase enzyme producing linolenic acid from linolic acid is
deficient, therefore, a soybean oil of a decreased content of
linolenic acid is obtained.
Sequence CWU 1
1
1311356DNAsoybean (Glycine max) 1tatttgcatt gtattgatag cccctccatt
cccaagagta taaaactgca tcgaataata 60caagccacta ggcatgggtc tagcaaagga
aacaacaatg ggaggtagag gtcgtgtggc 120caaagtggaa gttcaaggga
agaagcctct ctcaagggtt ccaaacacaa agccaccatt 180cactgttggc
caactcaaga aagcaattcc accacactgc tttcagcgct ccctcctcac
240ttcattctcc tatgttgttt atgacctttc atttgccttc attttctaca
ttgccaccac 300ctactccacc tccttcctca acccttttcc ctcattgcat
ggccaatcta ttgggttctc 360caaggttgcc ttctcactgg tgtgtgggtg
attgctcacg agtgtggtca ccatgccttc 420agcaagtacc aatgggttga
tgatgttgtg ggtttgaccc ttcactcaac acttttagtc 480ccttatttct
catggaaaat aagccatcgc cgccatcact ccaacacagg ttcccttgac
540cgtgatgaag tgtttgtccc aaaaccaaaa tccaaagttg catggttttc
caagtactta 600aacaaccctc taggaagggc tgtttctctt ctcgtcacac
tcacaatagg gtggcctatg 660tatttagcct tcaatgtctc tggtagaccc
tatgatagtt ttgcaagcca ctaccaccct 720tatgctccca tatattctaa
ccgtgagagg cttctgatct atgtctctga tgttgctttg 780ttttctgtga
cttactctct ctaccgtgtt gcaaccctga aagggttggt ttggctgcta
840tgtgtttatg gggtgccttt gctcattgtg aacggttttc ttgtgactat
cacatatttg 900cagcacacac actttgcctt gcctcattac gattcatcag
aatgggactg gctgaaggga 960gctttggcaa ctatggacag agattatggg
attctgaaca aggtgtttca tcacataact 1020gatactcatg tggctcacca
tctcttctct acaatgccac attaccatgc aatggaggca 1080accaatgcaa
tcaagccaat attgggtgag tactaccaat ttgatgacac accattttac
1140aaggcactgt ggagagaagc gagagagtgc ctctatgtgg agccagatga
aggaacatcc 1200gagaagggcg tgtattggta caggaacaag tattgatgga
gcaaccaatg ggccatagtg 1260ggagttatgg aagttttgtc atgtattagt
acataattag tagaatgtta taaataagtg 1320gatttgccgc gtaatgactt
tgtgtgtatt gtgaaa 135621357DNAsoybean (Glycine max) 2tatttgcatt
gtattgatag cccctccatt cccaagagta taaaactgca tcgaataata 60caagccacta
ggcatgggtc tagcaaagga aacaacaatg ggaggtagag gtcgtgtggc
120caaagtggaa gttcaaggga agaagcctct ctcaagggtt ccaaacacaa
agccaccatt 180cactgttggc caactcaaga aagcaattcc accacactgc
tttcagcgct ccctcctcac 240ttcattctcc tatgttgttt atgacctttc
atttgccttc attttctaca ttgccaccac 300ctacttccac ctccttcctc
aacccttttc cctcattgca tggccaatct attgggttct 360ccaaggttgc
cttctcactg gtgtgtgggt gattgctcac gagtgtggtc accatgcctt
420cagcaagtac caatgggttg atgatgttgt gggtttgacc cttcactcaa
cacttttagt 480cccttatttc tcatggaaaa taagccatcg ccgccatcac
tccaacacag gttcccttga 540ccgtgatgaa gtgtttgtcc caaaaccaaa
atccaaagtt gcatggtttt ccaagtactt 600aaacaaccct ctaggaaggg
ctgtttctct tctcgtcaca ctcacaatag ggtggcctat 660gtatttagcc
ttcaatgtct ctggtagacc ctatgatagt tttgcaagcc actaccaccc
720ttatgctccc atatattcta accgtgagag gcttctgatc tatgtctctg
atgttgcttt 780gttttctgtg acttactctc tctaccgtgt tgcaaccctg
aaagggttgg tttggctgct 840atgtgtttat ggggtgcctt tgctcattgt
gaacggtttt cttgtgacta tcacatattt 900gcagcacaca cactttgcct
tgcctcatta cgattcatca gaatgggact ggctgaaggg 960agctttggca
actatggaca gagattatgg gattctgaac aaggtgtttc atcacataac
1020tgatactcat gtggctcacc atctcttctc tacaatgcca cattaccatg
caatggaggc 1080aaccaatgca atcaagccaa tattgggtga gtactaccaa
tttgatgaca caccatttta 1140caaggcactg tggagagaag cgagagagtg
cctctatgtg gagccagatg aaggaacatc 1200cgagaagggc gtgtattggt
acaggaacaa gtattgatgg agcaaccaat gggccatagt 1260gggagttatg
gaagttttgt catgtattag tacataatta gtagaatgtt ataaataagt
1320ggatttgccg cgtaatgact ttgtgtgtat tgtgaaa 135731405DNAsoybean
(Glycine max) 3tcttctaggt tattacgcac cacccaccac gtatccctga
aaagagagaa aaacacacta 60agccaaagcc aaagcagcaa tggttaaaga cacaaagcct
ttagcctatg ctgccaataa 120tggataccaa caaaagggtt cttcttttga
ttttgatcct agcgctcctc caccgtttaa 180gattgcagaa atcagagctt
caataccaaa acattgctgg gtcaagaatc catggagatc 240cctcagttat
gttctcaggg atgtgcttgt aattgctgca ttggtggctg cagcaattca
300cttcgacaac tggcttctct ggctaatcta ttgccccatt caaggcacaa
tgttctgggc 360tctctttgtt cttggacatg attgtggcca tggaagcttt
tcagatagcc ctttgctgaa 420tagcctggtg ggacacatct tgcattcctc
aattcttgtg ccataccatg gatggagaat 480tagccacaga actcaccatc
aaaaccatgg acacattgag aaggatgagt catgggttcc 540attaacagag
aagatttaca agaatctaga cagcatgaca agactcatta gattcactgt
600gccatttcca ttgtttgtgt atccaattta tttgttttca agaagccccg
gaaaggaagg 660ctctcacttc aatccctaca gcaatctgtt cccacccagt
gagagaaaag gaatagcaat 720atcaacactg tgttgggcta ccatgttttc
tctgcttatc tatctctcat tcataactag 780tccacttcta gtgctcaagc
tctatggaat tccatattgg atatttgtta tgtggctgga 840ctttgtcaca
tacttgcatc accatggtca ccaccagaaa ctgccttggt accgcggcaa
900ggaatggagt tatttaagag gtggcctcac cactgtggat cgtgactatg
gttggatcta 960taacattcac catgacattg gcacccatgt tatccaccat
cttttccccc aaattcctca 1020ttatcacctc gttgaagcga cacaagcagc
aaaaccagtt cttggagatt actaccgtga 1080gccagaaaga tctgcgccat
taccatttca tctaataaag tatttaattc agagtatgag 1140acaagaccac
ttcgtagtga cactggagat gttgtttatt atcagactga ttctctgctc
1200ctccactcgc aacgagactg agtttcaaac tttttgggtt attatttatt
ggattctagc 1260tactcaaatt actttttttt taatgttatg ttttttggag
tttaacgttt tctgaacaac 1320ttgcaaatta cttgcataga gagacatgga
atatttattt gaaattagta aggtagtaat 1380aataaatttt gaattgtcag tttca
140541376DNAsoybean (Glycine max) 4tcttctaggt tattacgcac cacccaccac
gtatccctga aaagagagaa aaacacacta 60agccaaagcc aaagcagcaa tggttaaaga
cacaaagcct ttagcctatg ctgctaataa 120tggataccaa caaggaagct
tttgatccca gtgctcctcc accgtttaag attgcagaaa 180tcagagttgc
aataccaaaa cattgctggg tcaagaatcc atggagatcc ctcagttatg
240ttctcaggga tgtgcttgta attgctgcat tgatggctgc tgcaagtcac
ttccacaact 300ggcttctctg gctaatctat tggcccattc aaggaacaat
gttctgggct ctgtttgttc 360ttggacatga ttgtggccat ggaagctttt
cagacagccc ttttctaaat agcctggtgg 420gacacatctt gcattcctca
attcttgtgc cataccatgg atggagaatt agccacagaa 480ctcaccatca
aaatcatgga cacattgaga aggatgaatc ctgggttcca ttaaccgaga
540agatttacaa gaatctagac aacatgacaa gacttgttag attcactgtg
ccatttccat 600tgtttgtgta tccaatttat ttgttctcaa gaagccccgg
aaaggaaggt tctcacttca 660atccctacag caatctgttc ccacccagtg
agagaaaggg aatagcaata tcaacactgt 720gttgggttac catgttttct
atgcttatct atctctcctt cataactagt ccagttctat 780tgctcaagct
ctatggaatt ccatattgga tatttgttat gtggctggac tttgtcacat
840acttgcatca ccatggtcat catcagaaac tgccttggta tcgcggcaag
gaatggagtt 900atttaagagg tggtctcaca actgtggatc gtgactatgg
ttggatcaat aacattcacc 960atgacattgg cacccatgtt atccatcatc
ttttccctca aattcctcat tatcacctcg 1020ttgaagcgac acaagcagca
aaatcagttc ttggagagta ttaccgtgag ccagaaagat 1080ctgcgccatt
accatttcat ctaataaagt atttaattca gagtatgaga caagaccaga
1140tgtggtttat tatcagactg attctctgct cctccactcg caacgagact
gagtttcaaa 1200ctttttgggt tattatttat tggattctag ctactcaaat
tacttttttt ttaatgttat 1260gttttttgga gtttaacgtt ttctgaacaa
cttgcaaatt acttgcatag agagacatgg 1320aatatttatt tgaaattagt
aaggtagtaa taataaattt tgaattgtca gtttca 137651395DNAsoybean
(Glycine max) 5tcttctaggt tattacgcac cacccaccac gtatccctga
aaagagagaa aaacacacta 60agccaaagcc aaagcagcaa tggttaaaga cacaaagcct
ttagcctatg ctgctaataa 120tggataccaa caaggaagct tttgatccca
gtgctcctcc accgtttaag attgcagaaa 180tcagagttgc aataccaaaa
cattgctggg tcaagaatcc atggagatcc ctcagttatg 240ttctcaggga
tgtgcttgta attgctgcat tgatggctgc tgcaagtcac ttccacaact
300ggcttctctg gctaatctat tggcccattc aaggaacaat gttctgggct
ctgtttgttc 360ttggacatga ttgtggccat ggaagctttt cagacagccc
ttttctaaat agcctggtgg 420gacacatctt gcattcctca attcttgtgc
cataccatgg atggagaatt agccacagaa 480ctcaccatca aaatcatgga
cacattgaga aggatgaatc ctgggttcca ttaaccgaga 540agatttacaa
gaatctagac aacatgacaa gacttgttag attcactgtg ccatttccat
600tgtttgtgta tccaatttat ttgttctcaa gaagccccgg aaaggaaggt
tctcacttca 660atccctacag caatctgttc ccacccagtg agagaaaggg
aatagcaata tcaacactgt 720gttgggttac catgttttct atgcttatct
atctctcctt cataactagt ccagttctat 780tgctcaagct ctatggaatt
ccatattgga tatttgttat gtggctggac tttgtcacat 840acttgcatca
ccatggtcat catcagaaac tgccttggta tcgcggcaag gaatggagtt
900atttaagagg tggtctcaca actgtggatc gtgactatgg ttggatcaat
aacattcacc 960atgacattgg cacccatgtt atccatcatc ttttccctca
aattcctcat tatcacctcg 1020ttgaagcgac acaagcagca aaatcagttc
ttggagagta ttaccgtgag ccagaaagat 1080ctgcgccatt accatttcat
ctaataaagt atttaattca gagtatgaga caagaccact 1140tcgtaagtga
cactggagat gtggtttatt atcagactga ttctctgctc ctccactcgc
1200aacgagactg agtttcaaac tttttgggtt attatttatt ggattctagc
tactcaaatt 1260actttttttt taatgttatg ttttttggag tttaacgttt
tctgaacaac ttgcaaatta 1320cttgcataga gagacatgga atatttattt
gaaattagta aggtagtaat aataaatttt 1380gaattgtcag tttca
1395625DNASoybean (glycine max) 6attgatagcc cctccgttcc caaga
25726DNAsoybean (glycine max) 7attgtgagtg tgacgagaag agaaac
26831DNAsoybean (glycine max) 8gggtctagca aaggaaacaa caatgggagg t
31926DNAsoybean (glycine max) 9atacacacaa agtcattacg cggcaa
261029DNAsoybean (glycine max) 10ttattacgca ccacccacca cgtatccct
291129DNAsoybean (glycine max) 11gttgcgagtg gaggagcaga gaatcagtc
291226DNAsoybean (glycine max) 12gtggatcgtg actatggttg gatcta
261319DNAsoybean (glycine max) 13cttcgtaagt gacactgga 19
* * * * *