JP2595636B2 - Gene encoding glutamine synthase - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a gene encoding glutamine synthase, which is a major enzyme for ammonia assimilation in plants, algae, microorganisms and the like.

[Problems to be solved by conventional technology and invention]

Glutamine synthase (GS) is used in plants, algae,
It is known that it is an important enzyme that plays a major route in ammonia assimilation in a wide range of organisms such as microorganisms. Also
GS is also a target enzyme for certain herbicides, and the inhibition of GS activity prevents the assimilation of ammonia.
Plant toxicity is thought to die due to the toxicity of the accumulated ammonia [Journal of Molecular and Applied Genet
ics, 2, 621,1984]. GS, which is such an important enzyme,
Biological and biochemical studies have been advanced, genes have been isolated from some organisms, and their nucleotide sequences have been determined. For example, kidney beans [J. Mol. Appl. Genet., 2 , 5,
89, 1984 and EMBO J., 5 , 1429, 1986], peas (EMBO
J., 6 , 1, 1987], Alfalfa [Mol. Gen. Genet., 20
3 , 221, 1986) and green algae (Nature, 306 , 337, 1983 and Ge
ne, 53 , 211, 1987].

In some plants, GS is closely related to photogermination, and is known to be induced by red light and also by gibberellin, which has a germination promoting effect. It has been shown to be regulated at the transcriptional level. But GS by light or gibberellin
The regulation mechanism of gene expression has not been fully elucidated at the molecular level.

(Means for Solving the Problems) Therefore, the present inventors have developed GS obtained from water-absorbed seeds of lettuce.
The nucleotide sequence of the cDNA clone was determined, it was identified that GS was encoded, and the present invention was completed.

That is, the gist of the present invention resides in a gene encoding glutamine synthetase represented by the nucleotide sequence shown in FIG.

 Hereinafter, the present invention will be described in detail.

In the present invention, lettuce seeds (eg, New York 515)
Improved) is red-lighted and germinated, and the seedling is ground with a Waring blender or the like, and phenol extraction, ethanol precipitation, etc., to obtain total RNA. Then, DNA and low molecular RNA are removed by lithium chloride treatment.
Since the target GS mRNA has a polyA moiety, the GS mRNA is subjected to an oligo (dT) cellulose column using a conventional method.
Poly (A ⁺ ) RNA (mRNA) is obtained.

Using this mRNA as a raw material, the modified Gubler-Hoffman method (Gen
e, 25 , 263, 1983), the first strand c
DNA is obtained, and then second-strand cDNA is synthesized by DNA polymerase to obtain double-stranded cDNA. Usually, after blunting the end with T4 DNA polymerase, a linker such as Eco RI is bound, for example, λg
Incorporate into t11 etc. and package into phage particles. The phage particles are transformed into E. coli, for example, E. coli Y108.
8.Infect E. coli DH5α strain and culture,
Obtain a cDNA library containing cDNA.

From the cDNA library, a clone that reacts with an anti-rabbit GS antibody obtained by immunizing a rabbit with the purified GS protein as an antigen is selected, and the cDNA incorporated in the clone is used as a probe and hybridized with the cDNA library. . Then, among those hybridized with the probe, those having a longer cDNA chain length are selected, introduced into a plasmid such as pUC9, and transformed into Escherichia coli, for example, Escherichia coli Y1090. The resulting transformant is cultured to obtain the integrated cDNA.

The nucleotide sequence of the cDNA thus obtained can be determined by, for example, the dideoxy method of Sanger et al. The nucleotide sequence of the gene encoding GS of the present invention is shown in FIG. 2 together with the corresponding amino acids.

(Effect of the Invention) The gene encoding the glutamine synthetase of the present invention is:
It is considered to be useful as a material for producing herbicide-tolerant plants, analyzing the mechanism of induction by light or gibberellin, or elucidating tissue specificity.

(Examples) Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited to the following Examples.

[1] Isolation of poly (A ⁺ ) RNA Lettuce seeds (New York 515 Improved)
After absorbing water for 0 to 20 hours, about 20 g of a young plant germinated at 25 to 30 ° C. for 4 to 10 days was added to 30 ml of an extraction buffer [0.1 M Tris-HCl (pH 9.0), 0.1 M sodium chloride, 10 mM EDTA and 1% SDS], 30 ml of phenol: chloroform: isoamyl alcohol (25: 24: 1) and 6 ml of β-mercaptoethanol in a Waring blender. The triturated liquid was centrifuged at 7,000 rpm for 10 minutes, and the upper aqueous layer was taken. An approximately equal amount of phenol: chloroform: isoamyl alcohol (25: 24: 1) was added, mixed well, and then centrifuged again. The aqueous layer was collected. This phenol extraction was repeated once more, and the final aqueous layer
Add 5M sodium chloride to a final concentration of 0.25M, add 2.5 volumes of ethanol and mix well.
Left at 0 ° C. overnight. After that, centrifugation was performed at 8,000 rpm for 20 minutes, and the obtained precipitate was suspended in 70% ethanol.
The precipitate was collected by centrifugation again, and dried under reduced pressure.

The thus obtained dried total nucleic acid sample was dissolved in 15 ml of TE buffer (10 mM Tris-HCl (pH 7.5) and 10 mM EDTA).
10 ml of 5M lithium chloride was added to a final concentration of 2M, and the mixture was left overnight at 4 ° C. By centrifugation at 12,000 rpm for 20 minutes,
The total RNA fraction from which DNA and low molecular weight RNA remaining in the supernatant were removed was collected as a precipitate. The obtained precipitate was suspended in 2M lithium chloride, and the precipitate was recovered by centrifugation again, washed with 70% ethanol, and dried under reduced pressure to obtain a total RNA sample.

4.5 ml of the buffer solution [10
mM Tris-HCl (pH 7.5), 10 mM EDTA and 0.2% SDS], treated at 68 ° C. for 3 minutes, and quenched in ice water.
After adding 0.5 ml of 5 M lithium chloride and removing insolubles by centrifugation, the supernatant was equilibrated with 0.5 M lithium chloride, 10 mM Tris-hydrochloric acid (pH 7.5), 10 mM EDTA and 0.2% SDS. dT) applied to a cellulose column. The column was washed with about 5 volumes of a buffer (0.5 M lithium chloride, 10 mM Tris-hydrochloric acid (pH 7.5), 10 mM EDTA and 0.2% SDS), and then washed with 10 mM
Tris-HCl (pH 7.5), 1mM EDTA and 0.05% SDS poly
(A ⁺ ) RAN was eluted. Add 2.5 volumes of 5 M lithium chloride: ethanol (1:24) to the poly (A ⁺ ) RNA eluate,
After standing at ℃ overnight, the mixture was centrifuged at 30,000 rpm for 30 minutes, and the obtained precipitate was washed with 70% ethanol and dried under reduced pressure to obtain poly (A ⁺ ) RNA.

[2] Preparation of cDNA library 5 μg of dry poly (A ⁺ ) RAN was added to 20 μl of a reaction solution [50 mM Tris-HCl (pH 8.9) 10 mM magnesium chloride, 140 mM potassium chloride, 1.25 mM dNTP, 2 μg oligo (dT) _12-18 , 1
2.5mM dithiothreitol and 8mM sodium pyrophosphate], and heated at 65 ° C for 5 minutes, then cooled to 43 ° C,
[Α- ³² P] dCTP (400 Ci / mmol) and 40 units
And reverse reaction at 43 ° C. for 1 hour.
NA was synthesized.

Next, 8 μl of 200 mM Tris-hydrochloric acid (pH
1.1), 1 M potassium chloride and 50 mM magnesium chloride solution were added to adjust the reaction pH to 7.3. 5
Add μg bovine serum albumin (BSA), 25unti DNA polymerase, and 0.8 unit of RNase H.
After adjusting the volume to 00 μl, the mixture was reacted at 16 ° C. for 2 hours to synthesize second strand cDNA.

Further, 9 units (unit) of T4 DNA polymerase was added to the reaction solution, and the mixture was reacted at 37 ° C. for 10 minutes to smooth the ends of the double-stranded cDNA. Then, the reaction was stopped by adding 10 μl each of 0.25 mM EDKTA and 10% SDS, and after performing phenol extraction, 100 μl of 4 M ammonium acetate and 400 μl of ethanol were added and mixed, followed by cooling at −80 ° C. for 20 minutes, followed by 37 minutes. The mixture was centrifuged at a temperature of ° C for several minutes to recover double-stranded cDNA. This ethanol precipitation was performed once again, and the precipitate was washed with 70% ethanol and dried under reduced pressure.

The cDNA having blunt-ended ends was dissolved in 20 µl of a buffer [50 mM Tris-HCl (pH 7.5), 1 mM EDTA and 5 mM dithiothreitol], and 2 µl of 100 µM S-adenosyl-L-methionine and 20 units (unit) were added. Eco RI methylase was added and reacted at 37 ° C. for 20 minutes to methylate the Eco RI site of cDNA. The reaction was stopped by heating at 70 ° C. for 10 minutes, phenol extraction and ethanol precipitation were performed, and collected by centrifugation. To this was added 10 μl of a reaction solution [50 mM Tris-hydrochloric acid (pH 7.5), 10 mM magnesium chloride, 10 mM dithiothreitol, 2 mM ATP, 450 ng Eco RI linker and 175 units of T4 DNA ligase]. After reacting for an hour, an Eco RI linker was added. The reaction was stopped by heating at 70 ° C. for 15 minutes, and 10 μl of 50 mM Tris-HCl (pH 7.
5) Add 10 mM magnesium sulfate and 20 mM sodium chloride solution and add 38 units of Eco RI to add 3
Reaction at 7 ° C for 4 hours with Eco RI binding sites at both ends
cDNA was obtained. After the reaction, DNA was recovered by phenol extraction and ethanol precipitation. This DNA was added to 20 μl of TE buffer [10
mM Tris-HCl (pH 7.5) and 10 mM EDTA].
% Polyacrylamide gel electrophoresis, 600b
A gel containing p or more DNA was cut out. DNA was recovered from the gel by electroelution.

After dissolving this cDNA in 5 μl of sterile water, 5 μl of a reaction solution [0.5 μg Eco RI digested λgt11 DNA, 30 mM Tris-HCl (pH 7.4), 10 mM dithiothreitol, 10 mM magnesium chloride, 1 mM ATP and 30 units (unit) ) T4 DNA ligase], and allowed to react at 14 ° C for 4 hours.
And ligation. Next, packaging into phage particles was performed using Gigapack Gold (manufactured by Toyobo Co., Ltd.). Infect E. coli Y1088 with this phage,
After forming a plaque on L agar medium, 10 mM Tris-hydrochloric acid (pH 7.5) and 10 mM magnesium chloride were added, and the mixture was allowed to stand at 4 ° C. overnight. The liquid was recovered, and the cells were removed by centrifugation. Then, the supernatant phage solution was stored as a cDNA library.

[3] Identification of GS cDNA clone A phage was infected from a cDNA library into Escherichia coli Y1090, grown on L agar medium, and cultured at 37 ° C for 3.5 hours on a 10 mM IPTG-treated nitrocellulose filter. After culturing, remove the filter and use Proto Blot (Promeg
a), and screening was performed using an anti-GS rabbit antibody as an antibody.
Got one.

Using the cDNA incorporated in this clone as a probe,
The cDNA library was hybridized, and a plasmid having a long cDNA chain incorporated in λgt11 was selected from the hybridized ones.
The plasmid pLGS1 containing the cDNA encoding was obtained. This plasmid is treated with Eco RI to cut out cDNA, and
After introduction into UC9, E. coli Y1090 was transformed. The target cDNA was obtained by growing the transformant, and the entire nucleotide sequence was determined by the dideoxy method of Sanger et al.

FIG. 1 shows the nucleotide sequence of pLGS1 of 1429 bp.
GS is encoded by the 089th position and consists of 358 amino acid residues. In the figures, amino acids are represented by "one-letter symbols" (published by Tokyo Chemical Dojinsha, "Biochemical Dictionary", p.1392, 1984).

[Brief description of the drawings]

FIG. 1 is a drawing showing the nucleotide sequence of pLGS1 and the amino acid sequence translated based on it. In FIG. 1, GS corresponding to 358 amino acid residues from the 16th to the 1089th is encoded. FIG. 2 is a drawing showing a base sequence and an amino acid sequence of a portion corresponding to GS.

Continuation of front page (56) References EMBO J. 5 (7) P. 1429- 1435 (1986) EMBO J .; 6 (1) P. 1-9 (1987) Plant Cell Physiol. 24 p. 1477-1483 (1983)

Claims (1)

(57) [Claims] 1. A gene encoding glutamine synthase represented by the following nucleotide sequence: