JPH0638770A - Gene coding cysteine synthetase - Google Patents

Abstract

PURPOSE:To provide a new gene useful for increasing the cysteine content in a plant. CONSTITUTION:This invention is a gene coding plant cysteine synthetase and this gene has a base sequence of, e.g. the formula. A corresponding mRNA is taken out from a plant and cDNA is obtained according to vector-primer method by Okayama-Berg.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a gene encoding a plant cysteine synthase, and more particularly to a gene encoding a cysteine synthase which is a sulfur-containing amino acid and an essential amino acid of a plant.

[0002]

BACKGROUND OF THE INVENTION Cysteine is one of sulfur-containing α-amino acids and is used as a raw material for various pharmaceuticals, food additives, cosmetics and the like. In plants and microorganisms, cysteine is produced from O-acetylcholine and hydrogen sulfide as cysteine synthase [EC 4.2.99.
8; O-acetyl serine sulfhydrylase, O- acetylserine (thiol) - by the action of the lyase] it is known to be biosynthesized, Sal an enzyme level
The product purified from monella typhimurium (J. Biol. Chem., 244, 241).
8-2427, 1969; Biol. Chem. ，
244, 2428-2439, 1969), and also Sal
monella typhimurium and Es
A gene derived from C. cherichia coli has been isolated and identified (J. Bacteriology, 170,
3150-3157, 1988). In addition, such cysteine synthase is used in biosynthesis of non-proteinogenic amino acids having a neuroexcitatory action (Phytochemistry, 2
5,2759-2763,1986), it was clarified that it is involved in detoxification and conjugation of certain pesticides (Phy.
tochemistry, 29, 2507-2508,
1990), and is also interested in its physiological activity.

On the other hand, in order to improve the quality of wool, it has been studied to increase the content of sulfur-containing amino acids such as cysteine in grass. That is, increasing the cysteine content in agricultural products by expressing such a cysteine synthase gene in plants is considered to be a promising breeding method in terms of enhancing nutritional value, feed efficiency and the like. For that purpose, information on the gene of cysteine synthase in plants is required, but the present situation is that the plant-derived gene has not been isolated yet.

[0004]

[Means for Solving the Problems] The inventors of the present invention have conducted a genetic engineering technique to obtain a plant-derived cysteine synthetase cDN.
Focusing on the cloning of A, the present invention has been achieved by first obtaining a DNA segment encoding a cysteine synthetase required for such cloning.

That is, the gist of the present invention lies in a gene encoding a plant cysteine synthase. Hereinafter, the present invention will be described in detail. The gene encoding the cysteine synthase of the present invention has a corresponding mRN from the plant body as a raw material.
A is taken out and is used by the Okayama-Berg vector primer method (Molecular and Cel).
Ruler Biology, 2 , 161-170, 1
982), the method of Gubler & Hoffman (Gene, 25 , 263, 1983), etc.
Get A.

[0006] The plant used as a raw material is not particularly limited,
It is preferable to use higher plants such as solanaceous plants, gramineous plants, legumes, and rhododendrons. In the present invention,
Spinach ( Spinasia ol)
eracea ) is particularly preferably used, and specific examples thereof include commercially available seeds, for example, seedlings obtained by germinating parade (manufactured by Sakata Co., Ltd.), Yuparo (manufactured by Kyowa Seed Co., Ltd.) and Oscar (manufactured by Takii Co., Ltd.). Hereafter, spinach is used as a raw material and the method of Gubler & Hoffman (Gen
e, 25 , 263, 1983) to obtain the gene encoding the cysteine synthetase of the present invention.
The details will be described.

First, the green leaves of spinach seedlings are ground with a Waring blender or the like, and total RNA is obtained by phenol extraction, ethanol precipitation, or the like. Next, lithium chloride treatment removes DNA and low molecular weight RNA. When the mRNA of the target cysteine synthase has a polyA portion, it is subjected to poly (A ⁺ ) RNA (mR
NA) can be obtained.

Using this mRNA as a raw material, first-strand cDNA is first obtained by reverse transcriptase, and then second-strand cDNA is synthesized by DNA polymerase to obtain double-stranded cDNA. Usually, after blunting the ends with T4 DNA polymerase, a linker such as Eco RI is ligated, for example, λg
It is incorporated into t10, 11, etc. and packaged in phage particles. This phage particle was used for Escherichia coli Y1088, DH
5α, NK3 strain, etc. were cultured and cultured to obtain various lengths of c.
A cDNA library containing DNA is obtained.

The cysteine synthetase cDNA clone is prepared by digesting cysteine synthetase with an appropriate protease to synthesize a DNA probe deduced from the partial amino acid sequence of the peptide fragment, and c
Plaque hybridization method from DNA library (Maniatis T, et al, Molec
ural Cloning A Laboratory
Manual, Cold Spring Harbo
r Laboratory, 1982, p353-36.
It can be screened by 1) or the like. Such a cDNA clone is NK lacking cysteine synthesizing ability.
E. coli, such as 3 strains, was cloned into the p
It can be assayed by transforming with an expression vector such as TV118N (manufactured by Takara) and confirming the growth in a cysteine-deficient medium.

Further, from the cDNA library, a clone which reacts with an anti-rabbit antibody obtained by immunizing a rabbit with the purified cysteine synthetase protein as an antigen is selected, and the cDNA incorporated in the clone is used as a probe. Hybridize with cDNA library. Then, of those hybridized with the probe, those having a long cDNA chain length are selected and introduced into a plasmid such as pUC19 to transform Escherichia coli Y1088 or the like. The resulting transformant may be cultured to obtain the integrated cDNA.

The cDNA thus obtained is, for example, Sa
Nger et al.'s deoxy method (Proc. Natl. Aca
d. Sci. USA, 74, 5463, 1977), the base sequence can be determined. An example of the amino acid sequence and the base sequence of the gene encoding the cysteine synthetase of the present invention is shown in SEQ ID NO: 1 in the sequence listing.

[0012]

By expressing the gene encoding the cysteine synthase of the present invention in a plant, it is expected that the cysteine content is increased and the nutritional value and feed value in crops are increased.

[0013]

EXAMPLES The present invention will be described in more detail with reference to examples below, but the invention is not limited thereto unless it exceeds the gist.

[1] Poly from spinach green leaves
Preparation of A ⁺ RNA Spinach parade species ( spinsia oler
acea ) 70 days after germination, 42 g of green leaves are treated with 150 ml of extraction buffer [0.1 M NaCl, 10 mM EDT].
A, 1% SDS, 0.1M Tris: HCl (pH
9.0)], 150 ml of phenol: chloroform:
Isoamyl alcohol (25: 24: 1) and 30 ml of β-mercaptoethanol were added, and the mixture was ground with a Waring blender at 4 ° C. The milled liquid was centrifuged at 7,000 rpm for 10 minutes, an approximately equal amount of phenol: chloroform: isoamyl alcohol (25: 24: 1) was added to the upper aqueous layer, and after mixing well, the aqueous layer was collected again by centrifugation. This phenol extraction was repeated once more, and 5M NaCl was added to the finally obtained aqueous layer to a final concentration of 0.25M.
Then, 2.5 times the amount of ethanol was added and mixed well, and the mixture was allowed to stand at -20 ° C overnight. 8000 rp
The precipitate obtained by centrifugation for 20 minutes at m. was suspended in 70% ethanol, recovered by centrifugation again, and dried under reduced pressure.

15 ml ET of the resulting dried total nucleic acid preparation
Buffer [10 mM Tris: HCl (pH 7.5),
10 mM EDTA], and add 5 M LiCl to 10
ml was added to give a final concentration of 2M and left overnight at 4 ° C. Total RN excluding DNA and low molecular weight RNA remaining in the supernatant after centrifugation at 12,000 rpm for 20 minutes
Fraction A was collected as a precipitate. Precipitation with 2M LiCl was repeated again, washed with 70% ethanol, and then dried under reduced pressure to obtain 57 mg of total RNA preparation.

10 mg of dried total RNA preparation was added to 4.5 ml of
10 mM Tris: HCl (pH 7.5), 10 mM
 Dissolve in EDTA, 0.2% SDS, 68 ℃ for 3 minutes
After the treatment, it was quenched in ice water. 0.5 ml of 5 ml
iCl was added and the supernatant obtained by removing insoluble matter by centrifugation was
0.5M LiCl, 10 mM Tris: HCl in advance
(PH 7.5), 10 mM EDTA, 0.2% SD
About 1 ml of oligo-dT cellulose equilibrated with S
It was applied to a column (manufactured by Amersham). About column
After washing with 5 volumes of the same buffer, 10 mM Tris: H
Cl (pH 7.5), 1 mM EDTA, 0.05%
Poly A adsorbed to the column by SDS ⁺Elute RNA
did. poly A⁺2.5 volumes of 5M in RNA eluate
 LiCl: ethanol (1:24) was added, -20 ° C.
I left it overnight. Centrifugation at 30,000 rpm for 30 minutes
The precipitate obtained in step 1 was washed with 70% ethanol and dried under reduced pressure.
Dried and poly A⁺96 μg of RNA was obtained.

[2] Spinach green leaf poly A ⁺ R
Construction of NA cDNA library p prepared from spinach green leaves obtained in [1] above
3 μg of oli A ⁺ RNA was added to 9 μl of 5 mM Tri.
The mixture was heat-treated in s: HCl (pH 8.3) at 65 ° C for 5 minutes and then cooled to 43 ° C. Next, 4 μl of single-stranded synthesis reaction buffer [50 mM Tris: HCl
(PH 8.3), 50 mM KCl, 10 mM MgC
l ₂ , 3 mM DTT], 2 μl of 1.25 mM dN
TP, 1.35 μg of oligo (dT) primer, and 9 units of human placental ribonuclease inhibitor (Wako Pure Chemical Industries, Ltd.) were added to make a total volume of 20 μl. R in this reaction solution
26 units of everse transcriptase (Seikagaku Corporation) were added and reacted at 43 ° C. for 45 minutes. After the reaction was completed, put the tube in ice water and single-stranded cD
The synthesis of NA was completed.

Next, 3 were added to the single-strand cDNA synthesis reaction solution.
7.5 μl of double-stranded synthesis reaction buffer [100 mM
Tris: HCl (pH 7.4), 25 mM KC
1, 10 mM MgCl ₂ , 1 mM DTT], E. coli ribonuclease H, E. coli DNA polymerase I 2
Three units were added to bring the total volume to 100 μl. This reaction solution was first reacted at 12 ° C. for 60 minutes, then at 22 ° C. for 60 minutes, further at 70 ° C. for 10 minutes, and then centrifuged in an Eppendorf centrifuge for several seconds. Return to the ice bath again T4
Add 6 units of DNA polymerase and incubate at 37 ℃ for 10
After reacting for 2 minutes, both ends of the double-stranded cDNA were equilibrated.
To complete the reaction, 10 μl of 0.25M EDTA (p
H8) and 10 μl of 10% SDS was added. The reaction solution is
Phenol extraction After ether extraction, 25 μl of 4M ammonium acetate and 100 μl of ethanol were added, mixed, cooled at −70 ° C. for 20 minutes, and then centrifuged by DN.
A was collected. By this series of reactions, about 1.4 μg
It was possible to synthesize the double-stranded cDNA of This double-stranded c
CDNA cloning system λgt 10 kit (Amersham) using 0.5 μg of DNA
Was used to prepare a cDNA library.

[3] Preparation of Synthetic DNA Probe One-stranded DNA sequence V812 (a) predicted from the amino acid sequences of two peptide fragments produced by V8 protease digestion of spinach cysteine synthase.
And V822 (a) were synthesized and the ends were labeled with ³² P to prepare a probe. Synthetic DNA probe V812 (a)
The nucleotide sequence of V822 (a) is shown in SEQ ID NO: 2 in the sequence listing.
The nucleotide sequence of is shown in SEQ ID NO: 3 in the sequence listing.

[4] cDNA by synthetic DNA probe
Screening of library Of the cDNA library obtained in [2] above, about 2 × 10 ⁵ independent phage plaques were subjected to plaque hybridization with the probe prepared in [3] above. The final wash of the filter is 6 × SSC,
Performed at 0.1% SDS and 47 ° C.
19 clones positive for both (a) and V822 (a) were screened. About 1.3 kb Eco R
Two clones λcs2 and λcs4 with I insert
And insert the insert into the plasmid vector pUC19.
And M13mp18 were subcloned into the Eco RI site.

[5] Determination of Nucleotide Sequence of Cysteine Synthase cDNA The nucleotide sequence of a clone containing inserts derived from λcs2 and λcs4 was determined in both forward and reverse directions by the dideoxy method of Sanger et al. The entire base sequence of the insert portion 1303bp of λcs4 is shown in SEQ ID NO: 1 in the sequence listing. In SEQ ID NO: 1, from the 163rd amino acid threonine to the 182nd amino acid glycine, and 7
From the 1st serine to the 90th alanine are shown the portions corresponding to the amino acid sequences used for probe preparation, which correspond to V812 (a) and V822 (a), respectively.

[6] Assay of Cysteine Synthase cDNA Clone It was examined whether the cDNA clone cloned this time surely encodes cysteine synthetase. Escherichia coli (NK3) deficient in the ability to synthesize cysteine has
Expression vector pTV118N incorporating NA clone
(Takara) was introduced, and it was confirmed that it grew in a cysteine-deficient medium, and thus it was found that the cysteine synthesis ability was restored.

From this, it was confirmed that this cDNA clone encodes cysteine synthetase.

[0024]

[Sequence list]

SEQ ID NO: 1 Sequence length: 1303 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Hypothetical: No Antisense No Origin Origin organism name: Spincia oleracea strain name : Parade TCAGTCTCGA TTCTTCTCAG ATTTCTATCT CTTTTAAGTA TATAACCCAA A ATG GTT 57 Met Val 1 GAG GAG AAG GCC TTC ATT GCT AAA GAT GTG ACT GAA TTG ATT GGG AAA 105 Glu Glu Lys Ala Phe Ile Ala Lys Asp Val Thr Gly Leu 15 ACG CCA TTG GTA TAT CTC AAC ACT GTC GCC GAT GGT TGT GTT GCT CGT 153 Thr Pro Leu Val Tyr Leu Asn Thr Val Ala Asp Gly Cys Val Ala Arg 20 25 30 GTT GCT GCA AAG CTG GAA GGA ATG GAA CCT TGC TCT AGT GTT AAA GAC 201 Val Ala Ala Lys Leu Glu Gly Met Glu Pro Cys Ser Ser Val Lys Asp 35 40 45 50 AGG ATT GGG TTC AGT ATG ATT ACT GAT GCT GAA AAA AGC GGG CTT ATT 249 Arg Ile Gly Phe Ser Met Ile Thr Asp Ala Glu Lys Ser Gly Leu Ile 55 60 65 ACA CCT GGA GAG AGT GTC CTG A TT GAG CCC ACC AGT GGA AAT ACT GGC 297 Thr Pro Gly Glu Ser Val Leu Ile Glu Pro Thr Ser Gly Asn Thr Gly 70 75 80 ATT GGA TTA GCC TTC ATC GCA GCA GCT AAA GGT TAC AAG CTC ATC ATT 345 Ile Gly Leu Ala Phe Ile Ala Ala Ala Lys Gly Tyr Lys Leu Ile Ile 85 90 95 ACG ATG CCA GCA TCA ATG AGT CTT GAG CGG AGG ACT ATT CTC AGG GCC 393 Thr Met Pro Ala Ser Met Ser Leu Glu Arg Arg Thr Ile Leu Arg Ala 100 105 110 TTT GGT GCT GAG CTT ATC CTT ACT GAT CCA GCA AAA GGT ATG AAA GGG 441 Phe Gly Ala Glu Leu Ile Leu Thr Asp Pro Ala Lys Gly Met Lys Gly 115 120 125 130 GCT GTT CAG AAG GCT GAG GAG ATC CGT GAC AAA ACT CCT AAT TCA TAT 489 Ala Val Gln Lys Ala Glu Glu Ile Arg Asp Lys Thr Pro Asn Ser Tyr 135 140 145 ATA CTA CAA CAG TTT GAA AAC CCT GCC AAC CCA AAG GTT CAT TAT GAA 537 Ile Leu Gln Gln Phe Glu Asn Pro Ala Asn Pro Lys Val His Tyr Glu 150 155 160 ACA ACT GGA CCA GAA ATT TGG AAA GGC ACA GGT GGA AAA ATT GAT ATA 585 Thr Thr Gly Pro Glu Ile Trp Lys Gly Thr Gly Gly Lys Ile Asp Ile 165 170 175 TTC GTC TCT GGA ATA GGG ACT GGA GGT ACA ATA ACA GGT GCA GGA AAA 633 Phe Val Ser Gly Ile Gly Thr Gly Gly Thr Ile Thr Gly Ala Gly Lys 180 185 190 TAC CTA AAG GAA CAA AAC CCG GAT GTT AAG CTA ATT GGC CTG GAA CCA 681 Tyr Leu Lys Glu Gln Asn Pro Asp Val Lys Leu Ile Gly Leu Glu Pro 195 200 205 210 GTG GAA AGT GCT GTA TTG TCT GGA GGA AAA CCT GGC CCA CAT AAG ATT 729 Val Glu Ser Ala Val Leu Ser Gly Gly Lys Pro Gly Pro His Lys Ile 215 220 225 CAA GGA CTT GGA GCT GGA TTC ATA CCT GGT GTT CTG GAT GTG AAT ATT 777 Gln Gly Leu Gly Ala Gly Phe Ile Pro Gly Val Leu Asp Val Asn Ile 230 235 240 ATC GAT GAA GTG GTT CAG ATA TCC AGT GAA GAA GAA TCT ATT GAA ATG GCC 825 Ile Asp Glu Val Val Gln Ile Ser Ser Glu Glu Ser Ile Glu Met Ala 245 250 255 AAA TTG CTC GCC CTC AAG GAA GGT CTA CTG GTT GGG ATT TCA TCT GGT 873 Lys Leu Leu Ala Leu Lys Glu Gly Leu Leu Val Gly Ile Ser Ser Gly 260 265 270 GCT GCT GCT GCC GCT GCC ATT AAA GTG GCA AAG AGG CCT GAA AAT GCT 921 Ala Ala Ala Ala Ala Ala Ile Lys Val Ala Lys Arg Pro Glu Asn Ala 275 280 285 290 GGA AAA CTC ATC GTC GCT GTC TTT CCC AGC TTT GGC GAA CGA TAT TTA 969 Gly Lys Leu Ile Val Ala Val Phe Pro Ser Phe Gly Glu Arg Tyr Leu 295 300 305 TCC TCG GTG TTG TTT GAT TCA GTG AGG AAG GAG GCA GAG AGC ATG GTT 1017 Ser Ser Val Leu Phe Asp Ser Val Arg Lys Glu Ala Glu Ser Met Val 310 315 320 ATT GAG TCC TAAG TTCGAGTTCT CCTTTAATGG TCTTGGATAC TTCACAGGCT 1070 Ile Glu Ser 325 TTGGTCTTTT CATGTGGTCT AAATCCTAGT TTTCTTCTGT TTTCCCTTTC TTTTCTCGTA 1130 GACATGCAAA TCGTGGCCCT TCTGTATTAG AACAATGTCA GTTGTGTCCT TGATGTTTGG 1190 TCATCATGCA CTGGCAACTA TGGTTTGTGA TGTATAAAAT ACTCTTTCAT GTTCTTCCAT 1250 TACATATTGT TGATGAGGGA TTTCATGTGA TAAAAAAAAA AAAAAAAAAA AAA 1303 SEQ ID NO: 2 Sequence length: 50 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Origin Biological name : Spinacia oleracea sequence EieiaieitiarutishiaieitiwaititiaishishiaishishiaijitiaishiCYTTCCAIATYTCIGGICCIGTIGT 50 SEQ ID NO: 3 length of sequence: 56 SEQ type: nucleic acid Number of strands: single strand Topology: linear sequence type: other nucleic acid synthetic DNA source organism name: Spinacia oleracea sequence JishiaijishiaieitiarueieiaijishishieijiaishishieieitiaishishiaijitiarutiTICCGCTGGTCGGYTCIATCAGIAC 56

Claims (2)

[Claims] 1. A gene encoding a plant cysteine synthase. 2. The gene according to claim 1, which is represented by the nucleotide sequence set forth in SEQ ID NO: 1.