JPH06321995A - Glucan elicitor receptor derived from soybean - Google Patents

Abstract

PURPOSE:To obtain the subject new compound having each specific molecular weight and partiah amino acid sequence, useful as a material for establishing the breeding technology for fungus-resistant plants, or e.g. as a raw material to be used for the research materials for elicitor-bound site and signal transmission. CONSTITUTION:Soybean seeds are cultured for a week in vermiculite and then subjected to hydroponics for 15 days; the resultant roots are harvested and frozen at -80 deg.C, and an iced buffer solution is added to the roots followed by homogenization; the resultant slurry is filtered and the filtrate is centrifuged; the supernatant is ultracentrifuged and the precipitate is suspended in an iced buffer solution and an amphoteric surfactant is added thereto followed by agitation, glucan elicitor receptor (ER) is then solubilized from a membrane fraction, and then it is ultracentrifuged to recover a supernatant, which is then dialyzed and then concentrated using an ultrafiltration membrane and purified by e.g. column chromatography, thus obtaining the objective protein playing a role as the aimed glucan elicitor receptor having partial amino acid sequences of respective formulas I, II and III.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a protein derived from a soybean root plasma membrane fraction and functioning as a glucan elicitor receptor (hereinafter referred to as "ER").
INDUSTRIAL APPLICABILITY The protein of the present invention is extremely useful as a material for establishing a technique for producing ER, and for establishing a technique for breeding mold-resistant plants using DNA encoding ER. Further, it can be used as a mine plate for preparing a pit body for ER, and the obtained pit body and DN encoding ER
The antisense DNA for A is used to study the ER elicitor binding site and signal transduction.

[0002]

2. Description of the Related Art It is known that after infection with phytopathogenic microorganisms, an antibacterial substance called phytoalexin is newly synthesized in plants (M. Yoshikawa, Nature, 257 (1978),
546). In addition, substances that induce a resistance reaction (synthetic accumulation of phytoalexin) in plants have been found from phytophthora infestans (NTKeen, Science, 187 (1975), 74), and these substances are collectively called elicitors. . In soybean, elicitors are polysaccharides made from glucose and are reported to be β-1,6- and β-1,3-linked glucans.
(JK Sharp, et al., J. Biol. Chem., 259 (1984), 11321, M.
Yoshikawa, Plant Cell Engineering, 1.2 (1990), 695). Phytoalexin, which plays an important role in the soybean mortality, is called glyceolin (M. Yoshikawa, et al., Physiol. Pl.
ant. Pathol., 12 (1978), 73). Phytophthora megasperma f. Sp. Glyci, a type of pathogenic filamentous fungus in soybean
It is considered that a specific receptor (ER) for glucan elicitor derived from nea) is a protein that plays an important role in the synthesis and accumulation of antibacterial substance glyceolin. Regarding the ER specific to this elicitor, the amino acid sequence has not yet been elucidated, and only its purification method has been shown (EGCosio, et a
l., FEBS, 264 (1990), 235, EGCosio, et al., Eur.J. Bioc
hem., 204 (1992), 1115, T. Frey, Phytochemistry, 32 (199
3), 543). The affinity column ligand used in these reports is an acid hydrolyzate of the cell wall of soybean blight, and the elicitor for activity measurement is hepter β-glucoside, whereas the present inventors used the ligand for the affinity column and The elicitor used for the activity measurement is an enzyme treatment (Zymolyase 100T) on the cell wall of soybean blight.
The glyceolin-inducing activity is about 15 times higher by weight and about 150 times higher by mole than the elicitor used in the reported purification method.

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to provide a glucan elicitor receptor useful for establishing a breeding technique for mold-resistant plants.

[0004]

The present invention has a molecular weight of about 70.
Kd is a protein that functions as a glucan elicitor receptor containing the following partial amino acid sequences. 1. Lys Asn Gly Asp Gln Gln Glu Tyr Ile His Pro Ty
r Leu Glu Gly 2. Ile Asp Gly Asp Leu Val Gly Val Val Gly Asp Se
r 3. Ala Tyr Ser Ile Val Gln Asp The protein of the present invention has a function as a receptor for a glucan elicitor derived from Phytophthora.
That is, this protein binds to a glucan elicitor generated when a microorganism of the genus Phytophthora enters the cell,
It has a function of instructing microsomes and nuclei to increase the content of phytoalexin in cells.

The protein of the present invention is present at a position showing a molecular weight of about 70 KD on polyacrylamide gel electrophoresis as shown in FIG. Further, it has the following amino acid sequence as a part thereof. 1. Lys Asn Gly Asp Gln Gln Glu Tyr Ile His Pro Ty
r Leu Glu Gly 2. Ile Asp Gly Asp Leu Val Gly Val Val Gly Asp Se
r 3. Ala Tyr Ser Ile Val Gln Asp These amino acid sequences were obtained by transferring purified ER to a PVDF membrane (Millipore) by electroblotting, digesting it with lysyl endopeptidase (AP-I), and then fragmenting it. Peptides were collected from the PVDF membrane, and then fractionated by reverse phase HPLC (μ-Bondasphere 5μC8), and then analyzed for each peak component by a gas phase protein sequencer (manufactured by Applied Biosystems). Were determined.

[0006] The protein of the present invention can be prepared, for example, by Cosio et al.
(EJB, 204 (1992), 1115) is obtained by partially modifying the method. That is, by homogenizing soybean roots, leaves, stems, etc., a membrane fraction was recovered from the obtained slurry, purified by ion exchange chromatography, and further purified by affinity chromatography using elicitor as a ligand. can get. The elicitor used at this time is not particularly limited as long as it is derived from a Phytophthora genus microorganism, but Phytophthora megasperm
Those derived from a f. sp. glycinea race 1 (ATCC34566) are preferred.

Next, the present invention will be described in detail with reference to examples. However, this embodiment does not limit the technical scope of the present invention.

[0008]

【Example】

1) Purification of ER derived from soybean roots Soybean (variety: Greenhomer) seeds (manufactured by Takayama Seed) were cultivated for 1 week in vermiculite, and then hydroponically cultivated for 15 days to harvest roots of about 40 kg wet weight. After freezing the harvested roots at -80 ℃, 1.25L of ice-cold buffer (25mM Tris-HCl pH7.0, 30mM MgCl ₂ , 2mM D) was added to the roots with a wet weight of 2kg.
ithiothreitol, 2.5 mM potassium metabisulfite, 1 mM PMS
F) was added and homogenized for 2 minutes with a Waring blender.

The obtained slurry was filtered through Miracloth (manufactured by Calbiochem), and the filtrate was centrifuged at 10,000 xg at 4 ° C for 15 minutes. The supernatant was ultracentrifuged at 100,000xg, 4 ° C for 20 minutes, and the precipitate was suspended in 160 ml of ice-cold buffer (25 mM Tris-HCl pH 7.4, 0.1 M sucrose, 5 mM MgCl ₂ , 1 mM PMSF, 5 mM EDTA). . In order to solubilize ER from the membrane fraction, a final concentration of 0.25
The amphoteric surfactant ZW3-12 (manufactured by Boehringer) was added so that the amount became 10%, and the mixture was stirred at 8 ° C for 30 minutes. Solubilized ER
In order to recover the protein, ultracentrifugation was performed at 100,000 xg, 4 ° C for 20 minutes, and the supernatant was recovered. 165 ml of the supernatant was added to the buffer solution (50 mM, Tris
-HCl pH 8.0, 0.2% ZW3-12, 4 ℃) After dialyzing 4 times with 2L,
To remove protease from the sample and stabilize ER, add 5 ml of Protrap (Takara Shuzo), slowly stir at 8 ° C for 30 minutes, and then centrifuge (1,000 xg, 4 ° C, 2 minutes) to collect the supernatant. did. 160 ml of the obtained supernatant was concentrated to about 50 ml using an ultrafiltration membrane YM-10 (manufactured by Amicon), and then A buffer (50 mM Tris-HCl pH 8.0, 0.1 M sucrose, 5 mM MgCl _{2) was} added. , 1mM PMSF, 5mM EDTA, 0.2% ZW3
-12,4 ° C).

Then, it was applied to Q-Sepharose HP 26/10 (Pharmacia) and subjected to a linear gradient of 0 to 1 M NaCl to elute ER. ER is NaCl
It was eluted at a concentration of around 0.45M. Active fraction in 3 with A buffer
After doubling the dilution, it was applied to MonoQ10 / 10 (Pharmacia) and a 0 to 1M NaCl linear gradient was applied to elute 8 ml of ER. ER was eluted near the NaCl concentration of 0.25M.

Finally, the following operations were performed to purify ER by affinity gel using elicitor as a ligand. In order to remove non-specific adsorption to the gel carrier, the active fraction was collected, stirred with about 33 mg of maltose-bound glass gel (bed volume about 100 μl) at 8 ° C for 1 hour, and the gel was centrifuged (1,000 rpm, 4 ° C, 2 minutes). ), And the supernatant was collected. About 17 mg of elicitor-bound glass gel (bed volume: about 50 μl) was added to 8 ml of the supernatant, and the mixture was slowly stirred at 8 ° C. overnight. The gel was collected by centrifugation (1,000 rpm, 4 ° C., 2 minutes) and washed twice with 2 bed volumes of A buffer. The gel-bound ER was recovered by washing 3 times with 4 bed volumes of 0.1% SDS. Table 1 shows the amount of protein and the amount of ER in each of the above purification steps.

[0012]

[Table 1] Protein amount and ER amount in each purification step (using soybean root and wet weight of about 40 kg as a starting material) ──────────────────────── ───────── Protein (mg) ER amount (pmol) ───────────────────────────────── Membrane fraction 17900 30 Soluble fraction 2000 214 Q-Sepharose 190 205 MonoQ 49 233 Maltose flow-through fraction 45 220 Eliciter elution fraction 0.004 45 ──────────────────── ──────────── Elicator-bound glass gel elution fraction (10 μl) and M
onoQ active fraction, maltose-bound glass gel flow-through fraction, gradient gel for electrophoresis, SDS-PAGE plate 10 /
Electrophoresis was performed using 20 (manufactured by Daiichi Pure Chemicals Co., Ltd.) and stained with silver stain (manufactured by Daiichi Pure Chemicals Co., Ltd.). This is shown in FIG. In the figure, 1 is the MonoQ active fraction, 2 is the maltose-bound glass gel flow-through fraction, and 3 is the elicitor-bound glass gel elution fraction. As shown in the figure, the band indicated by ER was detected at a molecular weight of about 70,000.

The fact that a protein having a molecular weight of about 70,000 has elicitor binding activity means that the photoaffinity reagent SA
Using a complex of SD (Pierce) and elicitor that was iodinated, a protein with a molecular weight of about 70,000 was iodinated, and SDS-PAGE of the membrane fraction was analyzed by PV.
Western blotting onto a DF membrane and incubation of the PVDF membrane with an iodinated elicitor indicated that the protein with a molecular weight of approximately 70,000 was iodinated.

According to the method described above, ER is obtained from roots having a wet weight of about 40 kg.
About 3 μg was purified. Next, the Eli used for purification of ER
Sitter, maltose-bonded glass gel, elicitor-bonded glass
About how to make lath gel and how to measure ER binding activity
Explain. How to make I elicitor Elicitor is N.T.Keen (Plant Physiol., 71 (1983), 460,
Plant Physiol., 71 (1983), 466)
Made Phytophthora megasperma f.sp. glyc
inea) Race 1 (ATCC34566) cell wall zymolyre
Freeze elicitor by treating with ZE100T (Kirin Brewery)
Then, adsorb Zymolyase 100T on CM-cellulose.
The resulting flow-through fraction was subjected to gel filtration G-75 (Pharma
(Manufactured by SIA) and collected a fraction having an average molecular weight of 10,000.
It was Glyceolin-inducing elicitor activity in the collected fractions
Is measured by the method of M. Yoshikawa (Nature, 257 (1978), 546).
According to. 8 μg elicitor soybean cotyledon
, 550 μg of glycerin was induced after 24 hours
Was done.II How to make maltose-bonded glass gel A.M.Jeffrey, et al., (Biochem.Biophys.Res.Commun., 62
(1975), 608) and the like. Mart
36 mg of glucose 120 mg and 6 g of Glss, Aminopropyl (manufactured by Sigma)
H₂Suspended in O 2 and stirred at room temperature overnight. 36 ml of ethanol
Immediately after that, 864 mg Sodium bo was added to 72 ml of ethanol.
Add a solution of rohydride and sonicate for 2 minutes
Then, the mixture was stirred at room temperature for 5 hours. 288 ml H₂Add O and chill with ice
And adjusted to pH 5.6 with acetic acid. Malteau that did not combine
The gel is removed to remove about 1.8 L of H₂Washed with O 2. Cleaning liquid
Maltose contained in J.H.
Quantification according to the method of Roe, (J. Biol. Chem., 212 (1955), 335)
Then, the amount of maltose contained in the washing solution
The combined maltose amount was estimated. As a result, 6g of Glass,
60 mg of maltose was combined with Aminopropyl.III How to make elicitor-bonded glass gel A.M.Jeffrey, et al., (Biochem.Biophys.Res.Commun., 62
(1975), 608) and the like. Elishi
37 mg and Glass, Aminopropyl 490 mg in 6 ml H₂Suspended in O
And stirred at room temperature overnight. Add 6 ml of ethanol and
Immediately after, 144 mg of Sodium borohydride was added to 12 ml of ethanol.
Add lysate, sonicate for 2 minutes, and leave for 5 hours
Stir at temperature. 48 ml H₂Add O, cool with ice, and add pH 5.6 with acetic acid.
Was prepared. Ansulon trial of elicitors that did not combine
The amount of maltose contained in the wash solution, which was quantified using a drug
From this, the amount of elicitor bound to the gel was estimated. That conclusion
Fruit, 490 mg of Glass, Aminopropyl, 34 mg of Eli
Sitter joined.IV ER binding activity assay The composite of elicitor and tyramine (manufactured by Tokyo Chemical Industry) is Jo
Method from ng-Joo Cheong (The Plant Cell, 3 (1991), 127)
Was synthesized according to. Next, the complex of elicitor and tyramine
Was iodinated with chloramine T. ER binding
The activity was measured in the following reaction solution. 50mM Tris-HCl pH7.4 0.1M Saccharose 5mM MgCl₂ 1mM PMSF 5mM EDTA Assay buffer (500μl)
500 μg or less), and incubate at 0 ° C for 2 hours
7.0 nM iodinated elicitor tyramine complex
(70Ci / mmol) was added and incubated at 4 ° C for 2 hours.
It was Whatman GF / B (0.3% polyethyleneimine aqueous solution
The reaction solution was filtered for 1 hour or more), then 5 ml
Ice-cold buffer (10 mM Tris-HCl pH 7.0, 1 M NaCl, 10 mM M
gCl₂) 3 times, and the radioactivity remaining on the membrane was
Measured with a counter. Excludes the effects of non-specific binding
Therefore, add 17 μM elicitor to the same sample and
Incubate for 2 hours at 0 ° C after suspending in the buffer solution
Then perform the same operation below and insert the obtained count.
Count by specific binding rather than subtraction
It was 2) Analysis of fragmented peptides of elicitor receptor Peptide digestion of elicitor receptor was performed by protease digestion.
Cide fragmentation and Iwamatsu's method (Akihiko Iwamatsu, Biochemistry, 63
(1991), 139; A. Iwamatsu, Electorophoresis, 13
(1992), 142).
Determined the columns. Using the method described above and using a root with a wet weight of about 40 kg,
The licitor receptor was purified and the resulting sample was sent
Concentrate to approximately 100 μl with Con-30 (Amicon), 10-20%
Acrylamide SDS electrophoresis was performed and PVDF membrane (millimeter
Electroblotting to Pore (Sartrius)
Made). Transfer the sample and transfer the film to 0.1% Ponceau S / 1% vinegar.
Stain with acid and cut out the main band with a molecular weight of about 70,000
And decolorized with 0.5 mM NaOH. This is reduced S-carboxyme
Chill and ferment lysyl endopeptidase (AP-I)
Add to the base: substrate (mol: mol) ratio of 1: 100, and add 30
The reaction was carried out at ℃ for 16 hours. The fragmented peptide generated is μ
-Bondasphere 5μC 8-300Å (2.1x150mm, Waters) Color
With a linear gradient of 2-50% Solvent B
Elution was performed for 30 minutes at a flow rate of 0.25 ml / min (Solvent A: 0.05%
TFA in Water, Solvent B: 0.02% TFA in 2-propanol: ac
etonitrile = 7: 3 (v / v)). The eluted peptide is 214n
Each peak is detected manually by absorption at m
Collected. Each collected peak is a gas phase protein
Sequencer (Applied Biosystems Model 470A)
Analyzed. Fragmentation peaks analyzed from each peak
The peptide amino acid sequence was determined as follows.

1. Lys Asn Gly Asp Gln Gln Glu Tyr Il
e His Pro Tyr Leu Glu Gly 2. Ile Asp Gly Asp Leu Val Gly Val Val Gly Asp Se
r 3. Ala Tyr Ser Ile Val Gln Asp

[0016]

INDUSTRIAL APPLICABILITY In the present invention, ER was purified from the soybean root membrane fraction and digested with protease to determine the amino acid sequence of the obtained fragmented peptide. Thus, the determination of the amino acid sequence of a peptide derived from ER is the first in the world, and the present invention is extremely useful as a material for establishing a breeding technology for plants and the like using DNA encoding ER. Further, antisense DNA that can be used as a well for preparing a body for ER and is designed based on the information of the body and DNA encoding ER is a binding of elicitor to a receptor. Used to study site and receptor signaling. Therefore, the present invention is extremely useful industrially.

[Brief description of drawings]

FIG. 1 shows a polyacrylamide gel electrophoresis pattern.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Reference number within the agency FI Technical display location C07K 99:00 (72) Inventor Masaaki Yoshikawa 12-24 Kita-ku Nishi Kita-ku, Sapporo, Hokkaido 1-7 -A-901

Claims (1)

[Claims] 1. A protein which has a molecular weight of about 70 Kd and functions as a glucan elicitor receptor containing the following partial amino acid sequence. 1. Lys Asn Gly Asp Gln Gln Glu Tyr Ile His Pro Ty
r Leu Glu Gly 2. Ile Asp Gly Asp Leu Val Gly Val Val Gly Asp Se
r 3. Ala Tyr Ser Ile Val Gln Asp